Methods of acute restoration of vascular compliance

ABSTRACT

Disclosed herein is a compound for use in a composition applied to a blood vessel, wherein the compound softens and/or disrupts the crystalline matrix of calcified plaque, as well as acutely restoring the vascular compliance at the treatment site of the blood vessel, while maintaining luminal gain during angioplasty. Methods of treatment comprising applying the disclosed composition are also disclosed. Plaque-softening compounds are also disclosed.

DESCRIPTION OF THE INVENTION Field of the Invention

The present invention is directed to compounds that may disrupt the hardand crystalline structure of plaque. These compounds may be used in acomposition to soften plaque. Methods of use of the disclosed compoundsand/or compositions are also disclosed.

BACKGROUND OF THE INVENTION

Vascular plaque causes several medical conditions, including but notlimited to, coronary artery disease, carotid artery disease, andperipheral artery disease.

Atherogenesis is the developmental process of atheromatous plaques. Thebuild-up of an atheromatous plaque is a slow process, developed over aperiod of several years through a complex series of cellular eventsoccurring within the arterial wall, and in response to a variety oflocal vascular circulating factors. Atheromatous plaques form in thearterial tunica intima, a region of the vessel wall located between theendothelium and the tunica media. The bulk of these lesions are made ofexcess fat, collagen, and elastin. At first, as the plaques grow, onlywall thickening occurs without any significant narrowing. Stenosis is alate event, which may never occur and is often the result of repeatedplaque rupture and healing responses, not just the atheroscleroticprocess by itself. Such vascular stenoses are alternatively referred toas vascular lesions.

Intracellular microcalcifications form within vascular smooth musclecells of the surrounding muscular layer, specifically in the musclecells adjacent to the atheromas. In time, as cells die, this leads toextracellular calcium deposits between the muscular wall and outerportion of the atheromatous plaques. The outer, older portions of theplaque become more calcific, less metabolically active and morephysically rigid over time.

Two plaque types can be distinguished:

The fibro-lipid (fibro-fatty) plaque is characterized by an accumulationof lipid-laden cells underneath the intima of the arteries, typicallywithout narrowing the lumen due to compensatory expansion of thebounding muscular layer of the artery wall. Beneath the endotheliumthere is a “fibrous cap” covering the atheromatous “core” of the plaque.The core consists of lipid-laden cells (macrophages and smooth musclecells) with elevated tissue cholesterol and cholesterol ester content,fibrin, proteoglycans, collagen, elastin, and cellular debris. Inadvanced plaques, the central core of the plaque usually containsextracellular cholesterol deposits (released from dead cells), whichform areas of cholesterol crystals with empty, needle-like clefts. Atthe periphery of the plaque are younger “foamy” cells and capillaries.These type of plaques are sometimes referred to as vulnerable plaques,and usually produce the most damage to the individual when they rupture,often leading to fatal myocardial infarction when present within thecoronary arteries.

The fibrous plaque is also localized under the intima, within the wallof the artery resulting in thickening and expansion of the wall and,sometimes, spotty localized narrowing of the lumen with some atrophy ofthe muscular layer. The fibrous plaque contains collagen fibers(eosinophilic), precipitates of calcium (hematoxylinophilic) and,rarely, lipid-laden cells.

Atheromas within the vessel wall are soft and fragile with littleelasticity. In addition, the calcification deposits between the outerportion of the atheroma and the muscular wall of the blood vessel, asthey progress, lead to a loss of elasticity and stiffening of the bloodvessel as a whole.

The calcification deposits, after they have become sufficientlyadvanced, are partially visible on coronary artery computed tomographyor electron beam tomography (EBT) as rings of increased radiographicdensity, forming halos around the outer edges of the atheromatousplaques, within the artery wall. On CT, >130 units on the Hounsfieldscale (some argue for 90 units) has been the radiographic densityusually accepted as clearly representing tissue calcification withinarteries. A carotid intima-media thickness scan (CIMT can be measured byB-mode ultrasonography) measurement has been recommended by the AmericanHeart Association as the most useful method to identify atherosclerosis.

Intravascular ultrasound (IVUS) and optical coherence tomography (OCT)are the current most sensitive intravascular methods for detecting andmeasuring more advanced atheroma within living individuals. However,these imaging systems are seldom used for assessment of atheroma in viewof their cost, which is not reimbursed in many medical environments, aswell as invasive risks.

Angiography, since the 1960s, has been the traditional way of evaluatingatheroma. However, angiography is only motion or still images of dyemixed with the blood within the arterial lumen and do not directlyvisualize atheroma. Rather, the wall of arteries, including atheromawith the arterial wall, generally remain invisible, with only limitedshadows which define their contoured boundaries based upon x-rayabsorption. The limited exception to this rule is that with veryadvanced atheroma, with extensive calcification within the wall, ahalo-like ring of radiodensity can be seen in older patient, especiallywhen arterial lumens are visualized end-on. On cine-floro, cardiologistsand radiologists typically look for these calcification shadows torecognize arteries before they inject any contrast agent duringangiograms.

Interventional vascular procedures, such as percutaneous transluminalangioplasty (PTA) for peripheral vascular disease and percutaneoustransluminal coronary angioplasty (PTCA) for coronary artery disease,are typically performed using an inflatable balloon dilatation catheterto restore increased luminal diameter at the vascular lesion. During atypical PTA procedure, the dilatation catheter is positioned within theblood vessel at the location of the narrowing caused by the lesion, andthe balloon is expanded with inflation fluid to dilate the vessel lumen.Following the dilatation, it is common to introduce a second ballooncatheter which carries and deploys an expandable metal stent whichserves to maintain vessel patency.

However, patients with calcified plaque present a much more difficultchallenge for intervention. Indeed, presentation of diffuse, calcifiedvascular plaque within coronary arteries is often one of the mostcritical exclusion criteria for PTCA patient candidates, and thesepatients are instead required to receive invasive coronary artery bypassgraft (CABG) surgery to alleviate the coronary blood flow deficiencies.On the other hand, patients presenting diffuse, calcified vascularplaque in their peripheral arteries and veins may still be eligible forPTA vascular intervention, but these patients typically require apreliminary interventional procedure involving plaque removal, such asatherectomy catheters.

In the event that an atherectomy procedure is required, theinterventional physician must first deploy an embolic protection device(EPD) within the vessel being treated at a location which is distal(i.e., downstream relative to blood flow) to the atherectomy treatmentsite. Despite the adjunctive use of such an EPD, plaque particulateswhich are dislodged by the atherectomy device can occasionally escapethe EPD and travel downstream within the vasculature causing a stroke,heart attack or otherwise permanently compromised distal vascular bloodflow. In any event, the use of atherectomy devices produces substantialtrauma to the blood vessel, and can produce serious complications suchas thrombosis, as well as poor vascular healing response leading topremature restenosis.

To the extent that the interventional physician performs a PTA procedurewithin a blood vessel containing a lesion formed of calcified plaque,dilating such a lesion is more likely to produce increased vasculardamage to the vascular tissue, such as microdissections of the vasculartissue.

Significant research efforts have been made over the past several yearsto better understand the complex mechanisms associated with thedevelopment of atherosclerotic vascular disease, as well as thosemechanisms associated with restenosis following interventionalrevascularization procedures such as balloon angioplasty of targetlesions within arteries, as well as the eventual onset of in-stenttissue ingrowth or restenosis (ISR) following stent implantation withcertain patients.

For the past few decades, it has been generally understood thatrestenosis in general, as well as ISR, are largely the result of scartissue formation provoked at the vascular treatment site during thevascular healing response, which was initiated by the traumatic,inflammatory injuries resulting from PTCA, PTA and stent implantation.While other factors, such as genetics, diet, exercise, smoking, diabetesand the like clearly constitute factors which strongly influence patientoutcomes relative to interventional treatment of vascular disease, theinterventional cardiovascular community has only recently begun toproperly understand the complex biomechanics associated withangioplasty, stenting and restenosis.

With the advent of bare-metal stents (BMS) being implanted duringarterial revascularization procedures, a significant reduction inrestenosis with patients previously treating only with balloon catheterangioplasty has been obtained. In fact, it has become quite common forBMS to be implanted during catheter-based revascularization of coronaryarterial disease (CAD), which is deployed during percutaneoustransluminal coronary angioplasty (PTCA). Implantation of BMS has morerecently also become a useful adjunct during revascularization fortreatment of peripheral arterial disease (PAD), which are deployedduring percutaneous transluminal angioplasty (PTA).

With the introduction of BMS implantation as an adjunct to both PTCA andPTA procedures, in order to improve patency outcomes (i.e., reduceincidence of restenosis), it has become ordinary clinical practice toplace BMS patients following stent implantation on a three to four monthregimen of platelet-inhibiting drugs, such as Clopidogrel (e.g.,marketed by Bristol-Myers Squibb and Sanofi under the trade namePLAVIX®). Clopidogrel is an oral, thienopyridine class antiplateletagent used to inhibit blood clot formation in critical arterialvasculature (e.e., coronary, cerebral and certain peripheral arteries).This prophylactic administration of anti-platelet-aggregating agents isgiven to patients receiving a stent implant in order to mitigate therisk of thrombus formation at the treatment site while the vessel ishealing. During this critical healing period of approximately 90-120days following stent implantation, the vessel will normally repopulatethe treatment site with healthy endothelial cells, which will eventuallycover over the struts of the metallic stent implant, thereby removingthe foreign object from direct exposure to the bloodstream and potentialrisk of thrombus formation.

However, ISR continued to affect a significant portion of patientsreceiving BMS, thus requiring repeated interventions. In the case ofPTCA, occasionally patient death results before re-intervention can bemade. While the consequence of restenosis following PTA is certainlyless severe than PTCA, it has nonetheless proven quite difficult tomaintain patency of certain peripheral vasculature, due to the diffusenature of peripheral lesions, as well as the tendency of such lesions tobecome moderately to severely calcified.

In response to the relatively high incidence of ISR of BMS implants,over the past 15 years drug-eluting stents (DES) have been used. DEScomprise essentially a coated BMS, such that the BMS metallic platformserves as a substrate over which drug-impregnated polymer coatings aredeposited. The polymer matrix contains one of several anti-proliferativeagents (e.g., sirolium, everolimus, paclitaxel, etc.), which graduallyare released from the polymer coating, ultimately interacting with thevascular tissue to inhibit the onset of neointimal proliferation, oftencharacterized as neointimal hyperplasia (NH). NH is characterized by anuncontrolled proliferation of smooth muscle cell ingrowth, whichultimately produces ISR. The polymer matrix serves to provide a gradualrelease of the anti-proliferative agent into the tissue at the desiredrelease kinetics of between 30-120 days, to inhibit NH while the vesselis proceeding through the most critical healing period.

Unfortunately, potentially-fatal complications have unexpectedly emergedwith a portion of DES patients, known as late-stage thrombosis (LST).This late-stage formation of thrombus can occur at anytime, ranging fromseveral weeks, to several months or even years after the implant of theDES, often causing sudden, fatal myocardial infarction (MI). Ofparticular concern is the fact that MI events will often occur with DESpatients without any advance warning signs, such as angina or shortnessof breath.

The apparent cause of LST with DES has been attributed to adversereactions between the vascular tissue and the drug-impregnated polymercoatings used to provide the desired 30-120 day release kinetics profilerequired for the various anti-proliferative drugs being administered(e.g., sirolimus, everolimus, paclitaxel, etc.) Apparently, this tissuereaction directly interferes with, or disrupts, the necessary healingresponse associated with recruitment of healthy endothelial cells ontothe surface of the implanted stent, in order to remove the exposed stentstruts from the blood stream and eliminating risk of thrombus formation.

As a result, it has become necessary for DES patients to continue withtheir regimen of platelet anti-aggregating medication for extendedperiods, perhaps years or even a lifetime, in order to prevent LST. Ofcourse, the chronic use of these platelet-inhibiting drugs is not onlyenormously expensive, in comparison to occasional repeatedrevascularizations with patients receiving only BMS implants, but thesemedications produce numerous undesirable medical side-effects.Additionally, patients receiving anti-platelet therapy cannot submit toeven simple surgical procedures, unless they temporarily suspend theirmedication, in order to avoid severe bleeding. Of course, by suspendingthe anti-platelet medication, the DES patient is immediately atincreased risk of LST. Thus, the DES dilemma is one where the effort toreduce the risk of ISR, and the need for occasional repeat angioplasty,has produced a potentially much more serious risk of LST. Of course, itis beyond debate that vascular stents, and especially coronary stents,simply cannot be explanted from the patient. So a need clearly exists toimprove this approach.

Consequently, drug-eluting balloons have only recently been introducedas an alternative approach to delivery of anti-proliferative agents,such as paclitaxel, to the vascular treatment site. Unfortunately, theresults to date have not been very encouraging, and it has proven quitechallenging to avoid loss of the drug from the surface of the coatedballoon during introduction through the vasculature to the treatmentsite. It has proven to be equally challenging to effectively transfer anappropriate amount of the drug from the balloon surface and onto thevessel wall. Again, a need clearly exists to improve this approach.

It is accordingly a primary object of the invention to provide acompound, in the form of a composition, to be administered to a patientin need thereof, wherein the compound will not only disrupt thecrystalline structure of the calcified plaque resulting in at least oneof a softening of the plaque, and an increase in lumen diameter, butwill also acutely restore the vascular treatment site to more normallevels of compliance or distensibility, while retaining luminal gain.

SUMMARY OF THE INVENTION

In accordance with the invention, there is disclosed a kit of parts foruse in restoring vascular compliance comprising: a compositioncomprising a 4-amino-1,8-naphthalimide; a delivery system for deliveryof the composition into a blood vessel; and an activating agent foractivating the composition after the composition has been applied to theblood vessel.

In another aspect, there is disclosed a method of restoring vascularcompliance in a diseased blood vessel, comprising: inserting a deliverysystem into the blood vessel; applying a bolus of a compositioncomprising a 4-amino-1,8-naphthalimide compound to the blood vessel;activating the composition with a sufficient amount of an activatingagent to restore the vascular compliance of the blood vessel.

In a further aspect, there is disclosed a method of inhibiting smoothmuscle cell proliferation in a diseased blood vessel, comprising:performing an interventional procedure on the diseased blood vessel thatinitiates smooth muscle cell proliferation in the diseased blood vessel;and applying a bolus of a composition comprising a4-amino-1,8-naphthalimide compound to the diseased blood vessel;

wherein the application of the composition inhibits the smooth musclecell proliferation in the diseased blood vessel.

There is disclosed a method of restoring vascular compliance of a bloodvessel, comprising: performing an interventional procedure on thediseased blood vessel that initiates smooth muscle cell proliferation inthe diseased blood vessel; and applying a bolus of a compositioncomprising a 4-amino-1,8-naphthalimide compound to the diseased bloodvessel in an amount sufficient for the composition to reach an immediatearea of the diseased blood vessel where the interventional procedure wasperformed as well as the surrounding areas; wherein the application ofthe composition to the immediate and surrounding areas of theinterventional procedure restores the vascular compliance withoutexhibiting any areas of mismatched vascular compliance.

There is also disclosed a method of acutely restoring vessel complianceto a level approaching normal limits, comprising: performing arevascularization procedure on the vascular treatment site; applying abolus of a composition comprising a 4-amino-1,8-naphthalimide compoundto the vascular treatment site in an amount such that the vasculartreatment site immediately re-establishes a vasomotion function toaccommodate pulsatile blood flow, while retaining the luminal gainachieved during the revascularization procedure; wherein the vesselcompliance of the vascular treatment site is acutely restored to a levelapproaching normal limits.

Further, there is disclosed a method for maintaining luminal gain of adiseased blood vessel, comprising: increasing a luminal gain of adiseased blood vessel using a dilatation device; applying a compositioncomprising a 4-amino-1,8-naphthalimide compound to the diseased bloodvessel having an increased luminal gain; activating a composition withan activating agent to release polyether functional groups; reinforcinga wall of the diseased blood vessel with the released polyetherfunctional groups; wherein the reinforced wall retains the luminal gainwithout compromising the vascular compliance of the diseased bloodvessel.

Additional objects and advantages of the invention will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the invention. Theobjects and advantages of the invention will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate one (several) embodiment(s) ofthe invention and together with the description, serve to explain theprinciples of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an exemplary reaction scheme for tethering apharmacological agent to a tissue, such as a blood vessel.

FIGS. 2a and 2b illustrate exemplary synthetic pathways forplaque-softening compounds tethered to pharmacological agents.

FIG. 3 illustrates a reaction scheme for the activation of anaphthalimide compound of the present invention.

FIGS. 4a-f are photos illustrating various aspects of the presentinvention. FIGS. 4a and b are photos of an isolated section of a bloodvessel comprising a plaque matrix. FIGS. 4c-d are photos of the sameblood vessel after it has been subjected to angioplasty. FIGS. 4e-f arephotos of the same artery after it has been treated with aplaque-softening compound.

FIGS. 5a-f are photos illustrating various aspects of the presentinvention. FIGS. 5a and b are photos of an isolated section of a bloodvessel comprising a plaque matrix. FIGS. 5c-d are photos of the sameblood vessel after it has been subjected to angioplasty. FIGS. 5e-f arephotos of the same artery after it has been treated with aplaque-softening compound.

FIGS. 6a-f are photos illustrating various aspects of the presentinvention. FIGS. 6a-c are photos of an isolated section of a bloodvessel comprising a plaque matrix. FIGS. 6d-f are photos of the sameblood vessel after it has been treated with a plaque-softening compound.

FIGS. 7a-e are photos illustrating various aspects of the presentinvention. FIG. 7a is a photo of an isolated section of a blood vesselcomprising a plaque matrix. FIGS. 7b-c are photos of the same bloodvessel after it has been subjected to angioplasty. FIGS. 7d-e are photosof the same artery after it has been treated with a plaque-softeningcompound.

FIG. 8 is an image captured on a microscope showing the various layersof a blood vessel and the crystalline plaque (black cells) locatedbetween the two media layers of tissue.

FIGS. 9a and 9b are photos of an isolated section of a blood vesselbefore and after it has been subjected to a plaque-softening compound ofthe present invention.

FIG. 10 is a photo of an untreated section of a blood vessel afterangioplasty and exhibiting a tissue dissection or fissure.

FIG. 11a is a photo of a section of artery being activated. FIG. 11b isa photo of a side-by-side comparison of an untreated section of a bloodvessel (on the left) with a treated section of a blood vessel (on theright).

FIG. 12 is a photo of a treated section of blood vessel after activationof a plague-softening compound.

FIG. 13 is a graph illustrating arterial compression data for roseBengal.

FIG. 14 is a graph illustrating the percent luminal gain in untreatedand treated carotid porcine arteries and untreated and treated femoralporcine arteries.

FIG. 15 is a graph illustrating the distensibility coefficient forNative, Untreated, and Treated arteries.

FIG. 16 is a graph illustrating the cross-sectional compliance forNative, Untreated, and Treated arteries.

FIG. 17 is a photo illustrating the buckling of various blood vessels at200 mmHg of pressure.

FIG. 18 is a graph illustrating the buckling of various native,untreated, and treated blood vessels.

DESCRIPTION OF THE EMBODIMENTS

Reference will now be made in detail to the present embodiment(s)(exemplary embodiments) of the invention, an example(s) of which is(are) illustrated in the accompanying drawings. Wherever possible, thesame reference numbers will be used throughout the drawings to refer tothe same or like parts.

Over the past 5-10 years, significant insights have evolved relative tothe various mechano-biological mechanisms, which are believed to existat both molecular and cellular levels (e.g., turnover of collagen), andwhich manifest at tissue and organ levels. Only within the past fewyears, however, have these new insights been reported, observing thatmechanical loads at tissue and organ levels (e.g., increased pulsepressure) are apparently sensed by molecular structure and result inaltered gene expression, to the effect that the body will self-regulateits associated tissues and organs to maintain a biologically preferredstate of “tensional homeostasis.”

In recent medical literature, for example, relatively sophisticatedmathematically-based models have been proposed to both quantify andinterrelate the various mechanisms of arterial remodeling which appearto predominate operating under specific circumstances such ashypertension, which ultimately leads to vessel lumen loss (i.e.,stenosis and restenosis). Humphrey J. Mechanisms of Arterial Remodelingin Hypertension: Coupled Role of Wall Shear and Intraluminal Stress.Journal of Hypertension. 2008; 52: 195-200, the disclosure of which ishereby incorporated by reference.

This article (Humphrey Paper) reports the following observations andunderlying theories, which largely align with the tenet of tensionalhomeostasis (TH), such as the following:

a) Focal adhesions in smooth muscle cells and fibroblasts tend toincrease in area in response to local increases in mechanical loading,so as to maintain the stress constant at a biologically preferred level;

b) Fibroblasts tend to increase the tractions that they exert on theextracellular matrix when external loads are decreased from abiologically preferred value;

c) Vascular smooth muscle cells tend to re-lengthen to their normal,preferred values when an arteriole is forced into a vasoconstrictedstate for an extended period;

d) Arteries tend to decrease in caliber (i.e., lumen size) in responseto extension-induced increases in axial stress;

e) Stress fibers within endothelial and vascular smooth muscle cellsappear to disassemble and then reassemble in a mechanically preferredmanner when perturbed from a normal value of mechanical stress orstrain; and

f) Although changes in cytoskeleton and integrins occur within minutes,changes at the cell-cell and cell-matrix level occur over hours, andthose at the vascular level occur over days to weeks or months.

It has been observed that the flow of blood within a vessel produces awall shear stress (WSS) at the interface of the intima of the vascularwall, and that varying levels of WSS potentially contribute to theproliferation of neointimal proliferation and increased neointimalthickness (NT) and eventual stenosis and/or restenosis followingangioplasty. Cellular mechanisms within the vasculature treatment site,such as membrane receptors, are highly sensitive to conditions ofreduced vascular flow, and activate intracellular signals which promotethe proliferation of neointimal tissue, leading to restenosis.

The analysis of the complex in vivo biomechanics of arterial disease(e.g., pulsatile blood flow, nonlinear anisotropic vascular wallproperties, etc.) render scientific quantification of thesemechano-biological mechanisms quite challenging. However, at a highlevel the normal human artery is subjected to three primary mechanicalstresses, namely:

a) Blood-flow induced wall shear stress (WSS) T_(w);

b) Blood-pressure induced circumferential wall stress σ_(e); and

c) Axial wall stress σ_(z).

Axial wall stress is attributed to the contribution of elastin in thevascular wall, which appears to arise during development and to persistinto maturity because of the relatively long half-life of elastin.

Based upon the foregoing, the mean values of these three components ofstress (i.e., forces acting over oriented areas) can be calculated asfollows:

T _(w)=4μQ/(πa ³)  (Equation 1)

σ_(θ) =Pa/h  (Equation 2)

σ_(z) =f/πh(2a+h)  (Equation 3)

With respect to Equations 1-3 cited above, the following parameters aredefined below:

μ is the blood viscosity;

Q is the mean volumetric flow rate;

a is the luminal radius;

h is the wall thickness in any pressurized configuration;

F is the transmural pressure (with low perivascular pressure); and

f is the axial force that maintains the axial “prestretch” of thevessel.

With respect to Equation 2, the importance of the thickness:lumen ratio(h/a) becomes self-evident, noting that h is total thickness, not merelyintimal-medial thickness. With respect to Equation 3, the importance ofwall cross-sectional area becomes evident, which is often reported withregard to “eutrophic” versus “hypertrophic” vascular remodeling.Finally, while the importance of axial stretch in hypertension wasrecognized at least 15 years ago, it has received surprisingly littleattention, most likely due to the inability to reasonably infer specificvalues in vivo. Large arteries appear to maintain these stresses nearhomeostatic values (e.g., on the order of 1.5 Pa for T_(w) and 100 kPafor both σ_(θ) and σ_(z) in specific arteries, where 1 kPa=7.5 mm Hg).

The Humphrey Paper proposes relating perturbed values of flow Q andpressure P to original values via Q=εQ_(θ) and P=P₀, where a subscript₀or superscript⁰ denotes original and ε and γ denote sustained percentchanges from original (e.g., γ=1.3 if P increases 30% from original).

While Equations (1)-(3) above suggest that if mean shear stress, as wellas mean circumferential stress, are restored via growth and remodelingprocesses, then specific morphological changes to large arteries canperhaps be mathematically modeled as follows:

Analysis Step No. 1:

If: T _(w)=4μεQ ₀/(πa ³)(perturbed state)  (Equation 4)

And: τ_(w) ⁰=4μQ ₀/(πa ₀ ³)(original state)  (Equation 5)

Then: τ_(w)=τ_(w) ⁰  (Equation 6)

Requires: 4μ(εQ ₀)/(πa ³)=4μQ ₀/(πa ₀ ³)  (Equation 7)

Therefore: a=ε ^(1/3) a ₀  (Equation 8)

Analysis Step No. 2:

If: σ₀=(γP ₀)(ε^(1/3) a ₀)/h(perturbed state)  (Equation 9)

And: σ_(θ) ⁰=(P ₀ a ₀)/h ₀(original state)  (Equation 10)

Then: σ₀=σ_(θ) ⁰  (Equation 11)

Requires: (γP ₀)(ε^(1/3) a ₀)/h=(P ₀ a ₀)/h ₀  (Equation 12)

Therefore: h=ε ^(1/3) γh ₀₌  (Equation 13)

Applying certain of the Equations 1-13 cited above, a 30% sustainedincrease in flow alone should produce a 9% increase in both caliber andwall thickness. In contrast, a 30% sustained increase in pressure aloneshould produce a 30% increase in thickness but no change in caliber. Inother words, a mean stress-mediated growth and remodeling response wouldrequire coordinated changes in luminal radius a and wall thickness hbased directly on the percentage of perturbations in hemodynamics fromthe original state.

There is a complex biological interplay associated with sustainedhemodynamic vascular changes in either flow or pressure, whicheffectively serve as the means by which lumen radius tends towardε^(1/3)a₀ and thickness tends toward ε^(1/3)γh₀.

If luminal radius and wall thickness are dictated by flow and pressure,then restoring axial wall stress σz, to its original value requires achange in axial force f, which typically would cause a change in length(e.g., possible vessel tortuosity).

The general assumption that all of the various responses to each of theabove-mentioned stresses must be considered together is reinforced byreal world observations, coupled with certain identifiable changesproduced at the cellular and tissue levels due to the influence ofchange in vascular pressure (e.g., cyclic circumferential stress orstrain) and vascular flow (i.e., WSS).

While most of the previous vascular research concerning arterial biologyand mechanics has been focused on endothelial and smooth muscle cells,there is increasing evidence that adventitial fibroblasts play asignificant role in vascular homeostasis, as well as in diseaseprogression.

Vascular mechano-biology requires consideration of these sustainedhemodynamic vascular changes in either flow or pressure, and the complexshort-term and long-term cellular responses which they provoke,including for example, altered proliferation, migration,differentiation, apoptosis, synthesis and degradation of matrix,cross-linking of matrix, integrin binding that governs cell-matrixinteractions, and cadherin activity that governs cell-cell interactions.

Medical literature has reported an ever-increasing sigmoidalrelationship between increased WSS (τ_(w)) and endothelial NO synthasemRNA, and a decreasing sigmoidal relationship between increased WSS(τ_(w)) and endothelin-1 (ET-1) mRNA.

A complex interplay with respect to changes invasodilator/vasoconstrictor responses appear to be regulated by variousvascular parameters, including:

a) Increases in WSS (τ_(w)) provoke increases in endothelial productionof NO, which in turn acts as an inhibitor of smooth muscle cellproliferation.

b) Increases in cyclic stretch provoke increases in endothelialproduction of ET-1.

c) Increases in cyclic strain provoke increases in endothelialproduction of endothelial NO synthase.

d) Increases in WSS (τ_(w)) provoke decreases in endothelial ET-1production, which decreases the influence that ET-1 would otherwise haveto promote smooth muscle cell proliferation and synthesis of collagen.

e) Because WSS and cyclic circumferential stretch can changesimultaneously in vivo, analysis of vasoaltered states is presentlyapproached by reliance on more simplified biomechanical models.

By way of example, the influence of local increases in blood flow abovenormal limits will now be discussed. As noted earlier, the endotheliumup-regulates endothelial NO synthase, and increases its production of NOin response to local increases in blood flow above normal limits, whichin turn causes the vascular wall to dilate in an effort to restore WSS(i) toward the original levels. Once flow returns to normal levels, NOproduction returns to normal, and the vessel regains its originalcaliber according to a normal vasoactive response.

In the event, however, that the local increase in flow is sustained,such as during vigorous exercise, the increased production of NO enablescell and matrix reorganization or turnover to occur in the dilatedstate. Hence, combined WSS (τ_(w)) and intramural stress-mediated growthand remodeling in a sustained vasodilated state allows the vascular wallto become entrenched at a larger radius and wall thickness. Assumingblood-flow induced WSS (τ_(w)) eventually normalizes, NO productionlevels return to normal, and the biomechanics are reset to tensionalhomeostasis.

By way of alternate example, the influence of local increases in bloodpressure above normal limits will now be discussed. While large arteriesare nearly elastic, and thus distensible, an initial local increase inblood pressure tends to increase the luminal radius and isochoricallydecrease thickness. These changes serve to increase blood-pressureinduced circumferential wall stress σ_(θ), which sets into motion acascade of cellular-based stress-mediated responses, producesaccelerated growth of smooth muscle cells, and potentially endothelialand fibroblast cells, resulting in significant vascular remodeling.

Yet, the initial pressure-induced increase in caliber would likelydecrease blood-flow induced WSS (τ_(w)), which would then tend todecrease endothelial production of NO, while increasing production ofET-1 to restore WSS (τ_(w)) toward normal levels. Wall thickening thusoccurs in an initially constricted state at the original caliber viaincreases in smooth muscle (i.e., hyperplasia and/or hypertrophy) drivenby stress-mediated increases in platelet-derived growth factor (TGF-β)and extracellular matrix (e.g., particularly fibrillar collagens drivenby increases in TGF-β, connective tissue growth factors, etc.). Hence,the vascular wall again becomes entrenched within a vasoaltered state,with multiple stresses simultaneously playing important roles. Once thewall has thickened sufficiently to restore circumferential wall stressσ_(θ), toward original conditions (i.e., the increased ability of thewall to withstand the increased pressure), at a stable caliber and WSS(τ_(w)), the endothelium can return to its normal production of NO.

More specifically, the influence of WSS with respect to in-stentrestenosis (ISR) was studied and reported as a significant negativecorrelation between NT and WSS in a majority of the patients studied.Sanmartin M., et al. Influence of Sheer Stress on In-Stent Restenosis.Revista Espanola De Cardiologia. 2006; 59(1): 20-7, the disclosure ofwhich is hereby incorporated by reference. In particular, theproliferation of scar tissue produced within the arteries followingangioplasty, even without subsequent stenting, was triggered due todamage resulting from the mechanical stretching of the vessel wall(i.e., angioplasty), which exposed subendothelial structures to bloodflow and activation of a major thrombotic and inflammatory response. Theadditional process of stenting the revascularized vascular site,produced significant vessel damage and trauma to the vessel.

Moreover, the amount of neointimal proliferation was proportional to thedegree of damage caused, and local factors played a major role in thereparative response following angioplasty. The presence of the stentimplant at the luminal surface contributed to the increased neointimalresponse, (even the design of the stent can influence this response).While other factors, such as systemic variables (e.g., diabetes, geneticpolymorphisms, diet, exercise and the like) also contributed to the riskof restenosis, the role of fluid dynamics relative to neointimalproliferation was of real consequence to the progression of restenosis.Neointimal proliferation was highly correlated to relatively low WSSvalues at the vascular treatment site. The reduced WSS levels at eitherthe proximal and distal edges of the stent implant also corroboratedthat these local hemodynamics accounted for lumen loss due to theprogression of NT, which leads to in-stent restenosis (ISR).

In 2012, a relatively sophisticated series of vascular studies werereported regarding patients receiving bioresorbable vascular stent(BVS), formed of a polymer scaffolding comprising poly-lactide.Brugaletta S. et al. Vascular Compliance Changes of the Coronary VesselWall After Bioresorbable Vascular Scaffold Implantation in the Treatedand Adjacent Segments. Circulation Journal. 2012; 76: 1616-23, thedisclosure of which is hereby incorporated by reference herein.

The compliance of each vessel was calculated for each vessel segment(i.e., segment proximal and abutting the proximal edge of the implantedstent, segment directly supported by the stent scaffold, and the segmentdistal and abutting the distal edge of the implanted stent). Withrespect to the measured change in vessel compliance that was observed atthe moment immediately prior to implantation of the BVS stent, whencompared to the vessel compliance immediately after stent implantation,it was not surprising to observe the most significant difference wasobserved at the stented/scaffolded vessel segment. It was also notsurprising that compliance mismatch immediately following stentimplantation was most pronounced at the junction located between theproximal edge of the stented/scaffolded segment extending into theproximal segment, as well as the junction located between the distaledge of the stented/scaffolded segment extending into the distalsegment.

With respect to the patients treated by the BVS 1.0 stent, for example,a significant increase in vessel compliance was observed at the segmentthat was initially stented. The relatively pronounced increase in vesselcompliance observed at the late follow-up intervals was primarilyattributed to the gradual disappearance of the bioresorbable stentscaffolding.

It is believed that with the bioresorption of the BVS stent, the vesselsegment revascularized is eventually able to recover some of its nativevessel compliance and thereby better influence improved WSS levels tomitigate against restenosis. Additionally, with the eventualbioresorption of the polymer scaffolding, it is believed that theinitial compliance mismatch observed between the vessel segment stented,in comparison with the adjacent proximal and distal segments, mayeventually equalize, as well as a gradual dissipation of flowdisturbances and heterogeneous distribution of WSS once the stentimplant has fully resorbed.

In addition to needed improvements in revascularization of CAD,revascularization of the superficial femoral artery (SFA) also remainsamong the most challenging anatomy. The superficial femoral artery(SFA), for example, is certainly one of the most challenging peripheralvascular situations to revascularize. This is due to the fact that SFAlesions are typically quite diffuse, extending between 8-25 cm, andusually presenting calcified plaque. SFA patients often present withsymptoms of claudication, which is defined as reproducible ischemicmuscle pain, which often occurs during physical activity and is relievedafter rest. The claudicant experiences pain because of inadequate bloodflow.

Pulsatility is the fluctuation of blood pressure and blood flow velocityduring systole and diastole. As blood is pumped through the coronaryvessels, the vessel wall is exposed to two sets of forces, both of whichare critically important, namely: (a) WSS, which is the frictional forcegenerated on the intimal surface of the vessel wall as the blood flowsthrough it; (b) cyclic strain, which is the force generated by thestretching of the vessel wall during systole and is affected by vesseldistensibility (i.e., stretchability); and (c) the interplay of WSS andcyclic strain controls fundamental cell signaling which either leads inthe positive direction of atheroprotective/thromboresistant changes, orin the negative direction of disease progression and instability.

For instance, cyclic strain is known to stimulate eNOS gene regulation,and steady-state levels of prostacyclin are significantly increased ifthe WSS force is applied in a pulsatile fashion, in contrast to arelatively steady laminar flow rate.

Consequently, it is believed that cell signaling may become undesirablyaltered at locations where interactions between stent scaffolding andoverlying vascular tissue result in excessive inhibition of vasculardistensibility. These complex bio-feedback mechanisms which translatemechanical/fluid dynamics forces into pertinent chemical cell signals isreferred to as “mechanotransduction.”

Additionally, it is understood that applied mechanical strainpreferentially preserves collagen fibrils, and stretch of the vascularwall stimulates increased actin polymerization, activating the synthesisof smooth muscle-specific protein. Under such conditions, smooth musclecells preferentially maintain their contractile phenotype, while suchdifferentiation is lost in sites of vascular injury (i.e.,atherosclerosis or restenosis).

In summary, therefore, it is believed that the present inventionprovides a system and method to revascularize diseased arteries in amanner which acutely improves outcomes significantly. Among the mostimportant aspects of the present invention, the following uniqueadvantages are noted relative to vascular compliance:

The present invention utilizes a composition which is infused bycatheter into the vascular treatment site prior to vessel dilatation,and which penetrates both plaque and vascular tissues. It has beenobserved that the composition readily penetrates calcified plaque, andproduces a modified, pliable plaque which more readily responds to thesubsequent PTA dilatation.

The present invention provides a novel technology by which the dilatedvascular treatment site is stabilized for purposes of luminal gainretention, but without the need of implanting a stent-like prosthetic.The present invention achieves an immediate or acute restoration ofvascular compliance (i.e., pulsatility/distensibility) approachingnormal, healthy vascular behavior. Consequently, it is expected thatthis will facilitate a positive healing response, not only because thisprocedure enables retention of luminal gain without the need of atraumatic implantation of a prosthetic vascular stent scaffolding, butbecause the endothelium may experience an immediately favorable cellularenvironment in which positive chemical signals will follow (e.g., NO,prostacyclin, tissue plasminogen activators, thrombomodulin), therebypromoting healthy WSS levels, inhibiting smooth muscle cellproliferation, and minimizing inflammation.

The present invention improves the vascular healing response of diseasedvasculature in general, following revascularization procedures. This newtherapy provides an acute interventional function (i.e., during theinterventional revascularization procedure) to reinforce vascular tissue(e.g., natural vascular scaffolding comprising collagen and elastinfibrils disposed throughout the vascular wall structure of both arteriesand veins), as an adjunct to interventional procedures. The disclosedprocesses may have the ability to retain luminal gain followingangioplasty, without the necessity of implanting either a permanentmetallic stent, or a bioresorbable stent. Consequently, activation ofthe disclosed compound may not only provide an alternative approach toretention of luminal gain following angioplasty, without the necessityof implantation of a permanent or resorbable stent, but may serve toalso stabilize and fixate the remodeled plaque to prevent embolicevents.

This new therapy provides an acute interventional function (i.e., duringthe interventional revascularization procedure) to acutely restorevessel compliance at the treatment site. In stark contrast to thechronic difficulties associated with permanent prosthetic stent-likeimplants, such as BMS and DES, the present invention can restore thevessel compliance at the revascularization treatment site.

Due to the unique nature of the present interventional therapy, it maynot be necessary to deploy any stent implant, either permanent orbioresorbable. By avoiding the necessity of implantation of a stent, asubstantial portion of the vascular trauma associated with stentdeployment, as well as the potential for ISR, can be entirely avoided.Contrary to implantable resorbable stents, which requires several monthsto fully bioresorb, using the present invention the treated vessel canimmediately commence its healing response.

Moreover, since the revascularization procedure using the presentinvention virtually eliminates any need for an atherectomy procedure toprepare the vessel for angioplasty and stenting, as well as eliminatingthe need for stent implantation, a more prompt and improved healingresponse can be expected due to the elimination of the majority ofvascular trauma using conventional revascularization techniques.

For many of the reasons stated above, it may become completelyunnecessary to administer various pharmaceutically active agents, suchas anti-proliferatives, to manage the inflammatory response resultingfrom stent implantation.

Finally, with the prospect of eliminating prosthetic stent implants, andaccomplishing revascularization with substantially reduced vasculartrauma, the possibility has finally arisen that patients might be ableto safely discontinue the anti-platelet regimen within the a period ofonly 90 days following revascularization.

The present invention is directed to a naphthalimide compound comprisinga solubilizing functional group. Without being bound to any particulartheory, it is hypothesized that the naphthalimide compound may disruptthe crystal structure of the inorganic portion of the atheromatousplaque by drawing calcium from the crystal structure thereby weakeningand softening the plaque. This softening may facilitate additionalcompression of the plaque during treatment of the blood vessel therebyresulting in less damage to the blood vessel, which is known to be theresult of hard and sharp pieces of the calcified plaque disrupted byballoon angioplasty.

The naphthalimide compound may comprise a hydrophobic component thatallows the compound to penetrate the greasy portion of the plaque andaccess the calcium crystalline matrix. The disclosed naphthalimidecompound may have a higher affinity for calcium. The structure of thedisclosed compound may allow it specifically bind to calcium and otheralkali earth metals, allowing it to cross the membrane of cells, i.e.,can be used to control calcium concentration inside the cell.

The naphthalimide compound may be a 4-amino-1,8-naphthalimide compoundhaving a structure selected from the group consisting of:

wherein R, R′, and Q are each independently selected from the groupconsisting of straight-chain and branched chain alkyl groups having from2 to 200 carbons, optionally substituted with one or more ether, amideor amine groups; and wherein X is hydrogen. Naphthalimide compoundswhich may be used include those described in U.S. Pat. Nos. 5,235,045;5,565,551; 5,776,600; 5,917,045; 6,410,505; 7,514,399; and 8,242,114,the disclosures of all of which are hereby incorporated by reference.

In an aspect, R′ can be a substituted alkyl group, wherein the alkylgroup is substituted with heteroatoms, such as N, O, P, and S orhalogens, such as F, Br, CI, or I. In another aspect, R′ can include anamine, a carboxylate, a phosphate, and/or a sulfate.

In an aspect, Q is a polyethylene moiety. Moreover, Q can be a moietythat contains amines and carboxyl groups arranged in a fashionreminiscent of EDTA-like ligands, phosphate groups and/or organic acidsarranged in a fashion able to interact with calcium, or functionalmotifs able to interact with calcium such as luciferin.

In another aspect, Q is an acid or an alcohol, but can also be athioester, an organophosphorous ester, an anhydride, an amide, acarbamate, or an urea. In another aspect, the naphthalimide compound hasthe following structure:

or its geometrical isomers.

It is hypothesized that upon photoactivation the polyethylene moietylinking the two naphthalimides becomes an intermediate withphotoactivated terminal amines. This intermediate has an affinity forbinding to amino acid residues on biological molecules, and forms thelinkage via a condensation reaction. In particular, the naphthalimidemay have a higher affinity for linear protein structures such ascollagen or elongated elastin when compared to globular proteins, suchas albumin, because the constant twisting and turning of the backbonepulls the hydrogen bonds “out of phase”. Moreover, the dimer, shownabove, penetrates plaque easily and the diffusion rate is minimallyconstrained

The polyether moieties attached in the imide positions impart solubilityand the naphthalimide rings are for photoactivation. The solubilizingtails are also believed to mimic a crown ether effect present in knownchelating agents. Thus, it is believed that these solubilizing tailswould have the ability to penetrate the crystalline regions and disruptthe structure that makes the plaque hard and sharp. To be clear,however, there is a balancing act to be achieved between solubility anddiffusion that must be considered in formulating compounds for use inthe present invention.

Below are some additional compounds including a monomer, dimer, andtrimer of naphthalimide rings. Polymers and derivatives of the compoundsbelow are also contemplated.

The dimeric structure of the disclosed naphthalimide compound isdesigned to lie along the extended backbone of the collagen helix asshown below.

In considering alternative compounds for use in the disclosedcomposition, it is noted that as molecules get longer, at some point thestrong hydration associated with the terminal amines and the weakerhydration associated with the oxygen atoms will fail to overcome theinsolubility of the greasy naphthalimide and the naphthalimide will beinsoluble and therefore not useful. In particular, as the moleculebecomes longer and therefore more “greasy” it will more likely stick toone collagen molecule and not span the gap between two collagenmolecules. For this reason, linear trimers, such as exemplified above,may not be preferred.

In order to overcome the challenges of longer compounds, like a lineartrimer, a “capped” trimer, as shown below, comprising no terminal aminesand the polyethylene groups have been changed to polypropylene groupsmay be used. This molecule may have a smaller hydrodynamic radius and itmay be more hydrophobic. This may present an advantage in fasterdiffusion and an ability to penetrate plaque more effectively. Thedownside may be reduced water solubility.

A “starred timer” having two terminal amines, but not in a lineararrangement can be used as a compound of the invention. In addition, itis believed that this design increases the likelihood of linking withcollagen molecules. This behavior is not obvious from the monomer anddimer structures. Derivatives and polydisperse isomers of the compoundbelow are also contemplated.

The “starred” trimer is designed to overcome any intramolecular linksthat may form between the compound and the collagen matrix. The centergroup of that will become the linker retains the polyether functionalitybut the branched nature and additional methyl groups may reduce thetendency of the linker to hydrogen bond to the collagen backbone whileretaining the ability to associate with water. These characteristics mayincrease the likelihood of collagen intermolecular bonds and therebyincrease the effectiveness of the compound.

The “starred timer” extending along a collagen matrix is shown below.With this compound, the water solubility might be lower, the diffusionrate might be slower or the preferential localization with collagenmight be lower. Derivatives of the compound below are also contemplated.

Compounds other than the naphthalimide compounds disclosed above andtheir derivatives are also contemplated for use in a composition. Inparticular, compounds that possess functional groups that allow forwater solubility, increased tissue diffusion, and calcium solubilizationare considered useful for the present invention. Exemplary compounds,include but are not limited to, EDTA-like ligands, luciferin basedligands, polyether ligands, phosphate based ligands, and organic acids.

Ethylenediaminetetraacetic acid (EDTA) is a member of the polyaminocarboxylic acid family of ligands. EDTA binds to metals in a hexadentatefashion with an octahedral geometry. Numerous variants of this basicstructure have been used by chelating agents with various affinities fordifferent metals, such as calcium. Examples of compounds having a basicstructure similar to EDTA, include but are not limited to, ethyleneglycol tetraacetic acid (EGTA); diethylene triamine pentaacetic acid(DTPA); 1, 2-bis[o-aminophenoxy)ethane-N,N,N′N′-tetraacetic acid(BAPTA); andAmino-5-(3-dimethylamino-6-dimethylammonio-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N′,N′-tetraaceticacid. EDTA is completely hydrophilic and it is expected that it cannotpenetrate the greasy portion of plaque. Moreover, a relatively lowbinding constant (10.69 log K_(f)) between EDTA and calcium renders itunlikely that EDTA would be capable of removing calcium from plaque in ablood vessel.

Additional compounds, known for their use in fluorescence imaging, canbe used and comprise four carboxylic acid functional groups, such asFura 2 (C₂₉H₂₂N₃O₁₄ ⁵⁻), which binds to free intracellular calcium; Fura2-AM (C₄₄H₄₇N₃O₂₄); Fluo 3 (C₃₆H₃₀Cl₂N₂O₁₃); Fluo3-AM (C₅₁H₅₀Cl₂N₂O₂₃);Indo 1 (C₃₂H₃₁N₃O₁₂); Indo 1-AM (C₄₇H₅₁N₃O₂₂); Quin 2 (C₂₆H₂₃K₄N₃O₁₀);and Rhod 2-AM (C₅₂H₅₉ClN₄O₁₉). These compounds are available fromsuppliers such as Donjindo Molecular Technologies. The conjugatedaromatic group provides a fluorescence. The added aromatic group teachesthat changes can be made to the structure of the compound withoutcompromising the ability to soften plaque. Moreover, these groups areinherently greasy and therefore lipid soluble, which may provide theability to penetrate tissue.

Coelenterazine-WS, a luciferin based ligand, is an additional compoundthat can be used and is also supplied by Donjindo MolecularTechnologies.

A suitable polyether ligand for use as a compound in the presentinvention may be Calcium ionophore V—Selectophore®(10,19-Bis[(octadecylcarbamoyl)methoxyacetyl]-1,4,7,13,16-pentaoxa-10,19-diazacycloheneicosane),as shown below. This particular compound has long chain alkyl groupsattached that provide lipid solubility allowing the compound totransport calcium across cell membranes.

Variants of the crown ether motif have been reported by Gatto et al, II,J.A.C.S., vol. 106, No. 26, pp. 8240-8244 (1984) and Capel-Cuevas, etal, Talanta, Vol 78, pp. 1484-1488 (2009). These reports demonstratethat both the substituents as well as the constituent chalcogen can bevaried and still provide the ability to chelate calcium.

Phosphate based ligands, such as phosphonates or phosphonic acids, havebeen used to chelate calcium to prevent scale in water systems. Someexemplary compounds that may be useful as compounds in the presentinvention, include but are not limited to, etidronic acid (INN) or1-hydroxyethane 1,1-diphosphonic acid (HEDP);aminotris(methylenephosphonic acid) (ATMP); ethylenediaminetetra(methylene phosphonic acid) (EDTMP) (a phosphonate analog of EDTA);and diethylenetriamine penta(methylene phosphonic acid) (DTPMP).

Organic acids suitable for use as a compound of the present inventioninclude, but are not limited to, citric acid and dipicolinic acid(pyridine-2,6-dicarboxylic acid or PDC).

Common chelating agents include desfuroxamine mesylate (used for irontoxicity, dimercaprol (BAL) (lead, preferred for arsenic and mercury),DMSA—an analogue of dimercaprol (given for lead and arsenic),D-penicillamine (for lead, arsenic, or mercury), and calcium disodiumversante (CaNa2-EDTA). However, these compounds are generally used tochelate metals other than calcium, and may not have the requisitechemical structure to be water soluble and penetrate tissue, as neededby compounds of the present invention.

The disclosed naphthalimide compounds and alternative plaque-softeningcompounds can further comprise a tether linkage to a pharmacologicalagent. In particular, a pharmacological agent can be attached to the4-amino group of a naphthalimide compound through a tether. Uponactivation by an activating agent, the amino group containing the tetherand pharmacological agent can be controllably released in an active formthat will bond to tissues localizing the delivered pharmacological agenton the target tissue. To be clear, the pharmacological agent can bereleased over time depending on hydrolytic cleavage, photolysiscleavage, enzymatic cleavage, or a combination of hydrolytic cleavage,photolysis cleavage, and enzymatic cleavage of the pharmacologicalagent. The localization, solubility, and release profile of thepharmacological agent can be tailored by synthesis of the appropriatetether. The reaction scheme is exemplified in FIG. 1. The localization,solubility, and release profile of the pharmacological agent can betailored by synthesis of the appropriate tether.

FIG. 2A illustrates a synthetic pathway for the production of thenaphthalimide tethered to everolimus. FIG. 2B illustrates a syntheticpathway for the production of the naphthalimide tethered to paclitaxel.The reaction would be conducted with a DCC catalyst in organic solventwith purification to be complete on a silica gel column. The functionalgroup R (the tether) and the R′ (the tail optimized for localization andsolubility) will be chosen to impart the needed characteristics to themolecule. The tether R will control the rate of hydrolysis and thesolubility and R′ will control the water solubility of the compound.

An important consideration in the choice of R is the relationshipbetween the structure and the hydrolysis rate. Table 1 describes sixpossible tethers with anticipated hydrolysis rates that vary from slowto fast. The initial synthesis will center around conjugates with thetether derived from compounds 3 and 5. When used, R′ can be a polyether,JEFFAMINE® M-600 available from Huntsman. However, it is understood thatother R′ groups may be used to solubilize the resultant compound.

TABLE 1 Structure and anticipated hydrolysis rates of linkers. Source ofAnticipated Cleavage # Linker Structure Linker Rate 1         2            3         4

 

   

 

Linker available from Sigma-Aldrich Glycine   Linker available fromSigma-Aldrich 3-Amino- propionic acid     In situ generated       Linkeravailable from Sigma-Aldrich 3-amino-2- hydroxy- propionic acid Slow        Slow             Expected t_(1/2)~15 days Good for 30 day releaseIncreasing Rate Of Hydrolysis/Faster Drug Delivery

5

In situ generated Expected t_(1/2)~4 days Good for 7 day release 6

In situ generated Fast

Compounds 1 and 2 are expected to form simple ester tethers. Theseesters are expected to hydrolyze only slowly at physiological pH. In theunlikely situation that the local environment increases the hydrolysisrate to a level where the drug is delivered too quickly, these tetherscould be used to slow that rate.

Compound 3 has an electron withdrawing substituent near the carboxylicacid which will become part of the ester targeted for hydrolyticcleavage. The electron withdrawing group will speed the reaction fromcompounds 1 and 2. This tether will be synthesized as part of thenaphthalimide structure in situ before attachment to a pharmacologicalagent, such as everolimus. Literature reports show a hydrolysis ratethat is well suited to a 30 day delivery. The full structure of thisconjugate is shown below including the R′ tail.

Compound 4 has additional electron withdrawing characteristics whencompared to compound 3. This will provide a rate of hydrolysis that issomewhat faster than compound 3 and represents an example to tailor therelease rates.

Compound 5 has a structure that is even more susceptible to hydrolysisand can provide a faster rate of release. This tether can be synthesizedas part of the naphthalimide structure in situ before attachment to apharmacological agent. Literature reports show a hydrolysis rate that iswell suited to a 7 day delivery.

Of the compounds shown in Table 1, compound 6 will have the fastestrelease rate, likely too fast for a seven day delivery but represents apossible structure if the local environment stabilizes the ester andrelease rates are unexpectedly slow.

In an aspect, the nitrogen of the 4-amino naphthalimide connected to thetether can attach to the tissue after activation by an activating agent.The activating agent is selected from radiated energy, electromagneticenergy, laser, electric current, electrons, thermal neutrons, andchemicals. The tether/pharmacological agent will remain covalentlyattached to the tissue, likely collagen until such time that thecollagen is turned over. Hydrolysis of the ester linkage will result inthe release of the pharmacological agent in an unaltered state.

Localization of the compound with the tethered drug is controlledprimarily by the characteristics of the imide substituent. As anexample, polyether functional groups, which are hydrophilic, mayincrease the solubility of the pharmacological agent and may direct thelocalization to collagen rich regions, such as an arterial wall. Such ahydrophilic compound may easily enter the luminal side of an arterialwall and may penetrate into the media. Alternatively, a hydrophobiccompound may encounter a luminal barrier and be excluded from the media.

The pharmacological agent may be any agent comprising at least one ofalcohol functional groups. Alcohol functional groups on thepharmacological agent will be the target for attachment to thenaphthalimide compound via an ester linkage. Exemplary pharmacologicalagents comprising at least one alcohol functional group, include but arenot limited to, paclitaxel, everolimus, sirolimus, zotarolimus, andbiolimus. For example, both everolimus and sirolimus have a readilyavailable reactive alcohol in the 40-position that is a good synthetictarget for attachment. Similarly, both zotarolimus and biolimus have areadily available alcohol functional group in the 28-position.

Additional pharmacological agents that can be tethered to the compoundsof the present invention include anti-thrombogenic agents, such asheparin, and magnesium sulfate; antiproliferation agents, such aspaclitaxel and rapamycin; anticancer drugs; immunosuppressors;anti-infectives; antirheumatics; antithrombotic; HMG-CoA reductaseinhibitors; CETP inhibitors ACE inhibitors; calcium antagonists;antihyperlipidemics; integrin inhibitors; antiallergics; antioxidants;GPIIbIIIa antagonists; retinoids; carotenoids; lipid-level loweringmedicaments; DNA synthesis inhibitors; tyrosine kinase inhibitors;antiplatelets; antiinflammatories; tissue-derived biomaterials;interferons; monoclonal anti bodies; and NO production promoters.

Nonlimiting examples of the anticancer drugs include vincristine,vinblastine, vindesine, irinotecan, pirarubicin, doxorubicin,paclitaxel, docetaxel, mercaptopurine, and methotrexate.

Nonlimiting examples of the immunosuppressors include rapamycin and itsderivatives, tacrolimus, azathioprine, cyclosporine, cyclophosphamide,mycophenolate mofetil, gusperimus, and mizoribine.

Nonlimiting examples of the anti-infectives, include antibiotics,antifungal, antiviral, antimycobacteria, antiprotozoal,antihelmintics/antiparasitic, and vaccines. Antibiotics include but arenot limited to mitomycin, adriamycin, doxorubicin, actinomycin,daunorubicin, idarubicin, pirarubicin, aclarubicin, epirubicin,peplomycin, aminoglycosides, carbapenems, cephalosporins [1st-5thgeneration], aztreonam, fluoroquinolones, penicillins, macrolides,tetracyclines, monobactams, tigecycline, vancomycin, and zinostatinstimalamer. Antifungals include but are not limited to Amphotericin B,liposomal Amphotericin B, Lipid complex amphotericin B, flucytosine,nystatin, fluconazole, itraconazole, ketoconazole, posaconazole,voriconazole, terbinafine, caspofungin, micafungin, anidulafungin.Antivrials include but are not limited to acyclovir, adefovir,amantadine, cidofovir, entecavir, famciclovir, penciclovir, foscarnet,ganciclovir, interferon alpha, lamivudine, oseltamivir, ribavirinrimantadine, tenofovir, valacyclovir, valganciclovir, zanamivir,anti-HIV drugs. Anti-mycobacterials include but are not limited toethambutol, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine,para-aminosalicylic acid, streptomycin, amikacin.

Nonlimiting examples of the antirheumatics include methotrexate, sodiumthiomalate, penicillamine, lobenzarit, and DMARDs (disease modifyinganti-rheumatic drugs, such as abatacept, adalimumab, anakinra,etanercept, tocilizumab, infliximab, rituximab, chloroquine,sulfasalazine, gold salts).

Nonlimiting examples of the antithrombotics include heparin, lowmolecular weight heparins (fondaparinux, enoxaparin, dalteparin),aspirin, warfarin, clopidogrel, prasugrel, ticagrelor, rivaroxaban,dipyridamole, abciximab, antithrombotic preparations, ticlopidine, andhirudin.

Nonlimiting examples of the HMG-CoA reductase inhibitors includeserivastatin, serivastatin sodium, atorvastatin, nisvastatin,itavastatin, fluvastatin, fluvastatin sodium, simvastatin, rosuvastatin,and pravastatin.

Nonlimiting examples of the ACE inhibitors include quinapril,perindopril erbumine, trandolapril, cilazapril, temocapril, delapril,enalapril maleate, lisinopril, and captopril.

Nonlimiting examples of the calcium antagonists include hifedipine,nilvadipine, nicardipine, nifedipine, nimodipine, isradipine,felodipine, diltiazem, verapamil, benidipine, amlodipine, andnisoldipine.

Illustrative of the antihyperlipidemics is probucol, but may alsoinclude bile acid sequestrants, fibric acid derivatives, and statins.

Illustrative of the integrin inhibitors is AJM300.

Illustrative of the antiallergics is tranilast, but may also includeantihistamines, antileukotrienes, mast cell stabilizers, decongestants,and glucocorticoids.

Nonlimiting examples of the antioxidants include catechins,anthocyanine, proanthocyanidin, lycopene, and β-carotene. Among thecatechins, epigallocatechin gallate may be used.

Illustrative of the GPIIbIIIa antagonists is abciximab.

Illustrative of the retinoids is all-trans retinoic acid, but may alsoinclude Retinol, retinal, isotretinoin, alitretinoin, etretinate,acitretin, tazarotene, bexarotene, Adapalene.

Preferred examples of the flavonoids include epigallocatechin,anthocyanine, and proanthocyanidin.

Nonlimiting examples of the carotenoids include β-carotene and lycopene.

Illustrative of the lipid-level lowering medicaments is eicosapentaenoicacid including in combination with docosahexaenoic acid.

Illustrative of the DNA synthesis inhibitors are 5-FU, 6-mercaptopurine,6-thioguanine, allopurinol, capecitabine, cytarabine, fludarabine,gemcitabine, leucovorin, methotrexate, and pemetrexed.

Nonlimiting examples of the tyrosine kinase inhibitors include imatinib,sunitinib, gefitinib, erlotinib, genistein, tyrphostin, and erbstatin.

Nonlimiting examples of the antiplatelets include ticlopidine,cilostazol, and clopidogrel.

Nonlimiting examples of the antiinflammatories include steroids such asdexamethasone and prednisolone.

Nonlimiting examples of the tissue-derived biomaterials include EGF(epidermal growth factor), VEGF (vascular endothelial growth factor),HGF (hepatocyte growth factor), PDGF (platelet derived growth factor),and BFGF (basic fibrolast growth factor).

Illustrative of the interferons is interferon-γ1a.

Illustrative of the NO production promoters is L-arginine.

As to whether one of these pharmacological agents or a combination oftwo or more different ones should be used, a selection can be made asneeded depending on the case.

4-amino-1,8-naphthalimide compounds of the present invention can also belabeled. In an aspect, the compound is covalently bound to biotin viastandard DCC coupling methods, as an example. Alternative methods forlabeling a compound are known to those of ordinary skill in the art andare contemplated herein. The labeled compound will be easily detectableusing a fluorescent or enzymatic assay linked to streptavidin from astreptavidin horseradish peroxidase system.

It is also possible to radiolabel the compounds of the present inventionby the incorporation of labeled carbon, hydrogen, nitrogen, or oxygenduring the conversion of the pharmacological agent. Any suitableradiolabel or isotopic marker known in the art can be used, such ashydrogen, carbon, pnictogens, chalcogens, and halogens, etc. However, itis to be understood that the labeled compounds of the present inventionmust be safe for administration to humans.

The compounds disclosed herein can be dissolved in a solvent to form acomposition. In an example, the solvent can be phosphate buffered saline(PBS). Other suitable solvents include dimethylformamide and isopropylalcohol. In certain embodiments, the composition can optionally compriseone or more excipients, buffers, carriers, stabilizers, preservativesand/or bulking agents, and is suitable for administration to a patientto achieve a desired effect or result. The composition can be in anydesired form, including but not limited to a liquid, a solid, adispersion, a suspension, a hydrogel, a particle, a nanoparticle, a thinfilm, and and shaped structure.

The 4-amino-1,8-naphthalimide compounds can be present in thecomposition in a concentration from about 0.01 mg/mL to about 100 mg/mL,for example from about 0.1 mg/mL to about 50 mg/mL, and as a furtherexample from about 1 mg/mL to about 30 mg/mL. As a specific example, the4-amino-1,8-naphthalimide compound is present in a composition at aconcentration of 2 mg/mL.

The concentration of the compound, and optionally a tetheredpharmacological agent, can be chosen such that a therapeutic effect isachieved when released into a blood vessel. One of ordinary skill in theart would readily be able to determine the concentration of the compoundand/or the concentration of the pharmacological agent, in order toachieve the desired result.

The composition of the present invention may be provided in vials ofvarious sizes for ease of use. In particular, an 8 mL vial can be usedto hold 7 mL of the disclosed composition. The composition can bedispersed from the vial in one dose or is separate doses, for example afirst bolus of about 4 mL, followed by a second bolus of about 0.5 toabout 1.0 mL. In an aspect, a saline flush can occur between applicationof the first and second bolus. Additionally, the composition can becontained in a loadable cassette or a pre-loaded syringe.

It is envisioned that the plaque-softening compound and a compositioncomprising the compound could be stored in a freeze-dried form, whichcould be reconstituted with saline/PBS prior to use.

In an aspect, there is disclosed a method for using the4-amino-1,8-naphthalimide in a treatment zone of a blood vessel,optionally comprising a plaque matrix, the method comprising applying abolus of a composition comprising a 4-amino-1,8-naphthalimide compoundto the treatment zone of the blood vessel.

The composition disclosed herein can be applied to a blood vessel. In anaspect, a treatment zone of a blood vessel, such as an artery or vein,can be isolated. In another aspect, the composition is applied in anamount sufficient to provide a high systemic concentration. Thecomposition can be injected into the blood vessel. In an aspect, theblood vessel is the superficial femoral artery (SFA) and its collateralbranches. In another aspect, the composition of the present invention isapplied to an isolated section of a blood vessel for an extended periodof time, such as from about 1 second to about 1 hour, for example fromabout 1 minute to about 30 minutes, and for example from about 1 minuteto about 10 minutes. The amount of time can vary.

The compositions of the present invention can be used to soften plaque,which can improve problems associated with diabetes, and peripheralartery disease. The plaque lesions can vary in size. In an aspect, theplaque lesions range in length from about 1 to about 22 cm, for examplefrom about 4 to about 9 cm, and as a further example about 4 to about 7cm. The diameter of these plaque lesions can range from about 5 to about7 mm.

In the event the plaque lesion is longer than the device used to applythe 4-amino-1,8-naphthalimide compound in a single treatment, it isenvisioned that such longer plaque lesions can be treated in multiplestep treatments, wherein the length of the lesion, and the length of thedevice to apply to the composition are factors in determining how manytreatments may be needed to treat a lengthy plaque lesion.

In an aspect, the composition is delivered by a delivery systemcomprising an injection port and at least one balloon, such as atreatment balloon. A light fiber is in the lumen of the delivery systemand is designed to deliver blue light (i.e., 457 nm, for example 450-480nm, wavelength) at low power. The blue light activates the PEG-basedcomposition to cross-link with biomolecules of the vessel wall, such ascollagen.

Any delivery system, including catheter designs with at least oneballoon, can be used to deliver the plaque-softening composition to thetreatment area, e.g., blood vessel. An exemplary delivery system can befound in U.S. Provisional Application No. 61/679,591, entitled“Endovascular Multi-Balloon Catheters with Optical Diffuser forTreatment of Vascular Stenoses,”, as well as U.S. Pat. No. 8,242,114,the disclosures of each which are hereby incorporated by reference. Inan aspect, the distal end of a catheter can include a weeping balloonwith micropores to provide a gradual infusion to the treatment site ofthe disclosed composition.

In particular, the vessel can be prepared by initial dilatation usingangioplasty balloon to treat the stenotic region of diseased vessel(i.e., artery or vein). The composition is then injected between twoocclusion balloons which isolate the treated vessel wall and bathe thevascular tissue. A secondary dilatation balloon located between the twoocclusion balloons is inflated to restore the vessel lumen to thedesired diameter. The blue light is delivered to “activate” thecomposition. The activated composition cross-links with native collagenfibers and/or covalently bonds a tethered drug to the blood vessel wall.

When activated, the naphthalimide compounds of the present inventionhave a singlet charge transfer state, which does not produce singletoxygen. This is in contrast to singlet oxygen production, which isthrough triplet state sensitization. See Samanta, Ramachandram, Saoja,An investigation of the triplet state properties of 1,8-naphthalimides:a laser flash photolysis study, J. Photochem. Photobiol A; Chem, 101(1996), 29-32 (and references therein). The naphthalimide compounds ofthe present invention decay predominately by intramolecular chargetransfer state that leads to emission (C-T fluorescence). The lack ofoxygen dependence of the emission of the naphthalimide compoundindicates the charge transfer states are short lived.

The activation of the naphthalimide compound of the present invention isbelieved to follow the reaction scheme in FIG. 3. As illustrated in thereaction scheme, an exemplary dimer compound (I) is activated. The“linker” functional group (II) is released as well as two compounds(III). It is believed without being limited to any particular theory,that these reaction products (II) and (III) enter the systemiccirculation and is excreted by the kidneys.

Fluorescence studies can be used to demonstrate that the compositioncomprising the disclosed naphthalimide compound can penetrate the bloodvessel, and thus treatment over the entire area of the blood vessel canbe ensured.

There is disclosed herein a kit of parts for use in restoring vascularcompliance comprising: a composition comprising a4-amino-1,8-naphthalimide; a delivery system for delivery of thecomposition into a blood vessel; and an activating agent for activatingthe composition after the composition has been applied to the bloodvessel.

There is also disclosed a method of restoring vascular compliance in adiseased blood vessel, comprising: inserting a delivery system into theblood vessel; applying a bolus of a composition comprising a4-amino-1,8-naphthalimide compound to the blood vessel; activating thecomposition with a sufficient amount of an activating agent to restorethe vascular compliance of the blood vessel. The blood vessel can be anartery or a vein. The vascular compliance of the blood vessel isrestored to its native compliance within about 90 to about 120 daysafter the activating step

Prior to application of the composition, the diseased blood vessel doesnot exhibit native vascular compliance as a result of atherosclerosis, asurgical procedure, diabetes, hypertension, autoimmune disease,aneurysm, accident or injury, a minimally-invasive interventionprocedure, and repeated access by needle. The surgical procedure can beat least one of an endarterectomy, vascular graft implant, vascularanastomosis, and bypass graft. The minimally-invasive interventionalprocedure comprises at least one of PTA, PTCA, vascular stenting andatherectomy.

As a result of the activating step disclosed in the method above, theblood vessel retains its luminal gain from the insertion step without astent, wherein the stent is chosen from permanent stents and resorbablestents.

The method may further comprise, prior to insertion of the deliverysystem, a step of applying an initial bolus of a composition comprisinga 4-amino-1,8-naphthalmide compound to the blood vessel in order tosoften plaque present in the blood vessel.

There is also disclosed a method of inhibiting smooth muscle cellproliferation in a diseased blood vessel, comprising: performing aninterventional procedure on the diseased blood vessel that initiatessmooth muscle cell proliferation in the diseased blood vessel; andapplying a bolus of a composition comprising a 4-amino-1,8-naphthalimidecompound to the diseased blood vessel; wherein the application of thecomposition inhibits the smooth muscle cell proliferation in thediseased blood vessel.

The interventional procedure can be selected from the group consistingof repeated access by needle, endarterectomy, vascular graft implant,vascular anastomosis, bypass graft, PTA, PTCA, vascular stenting andatherectomy.

Due to the disclosed method, there is an increase in at least one of NOproduction, tissue plasminogen activators, and thrombomodulin.

There is also disclosed a method of restoring vascular compliance of ablood vessel, comprising: performing an interventional procedure on thediseased blood vessel that initiates smooth muscle cell proliferation inthe diseased blood vessel; and applying a bolus of a compositioncomprising a 4-amino-1,8-naphthalimide compound to the diseased bloodvessel in an amount sufficient for the composition to reach an immediatearea of the diseased blood vessel where the interventional procedure wasperformed as well as the surrounding areas; wherein the application ofthe composition to the immediate and surrounding areas of theinterventional procedure restores the vascular compliance withoutexhibiting any areas of mismatched vascular compliance. In an aspect,the immediate area of the diseased blood vessel wherein theinterventional procedure was performed retains a vascular compliancewhich is approximately equal to the vascular compliance of thesurrounding areas.

Further, there is disclosed a method of acutely restoring vesselcompliance to a level approaching normal limits, comprising performing arevascularization procedure on the vascular treatment site; applying abolus of a composition comprising a 4-amino-1,8-naphthalimide compoundto the vascular treatment site in an amount such that the vasculartreatment site immediately re-establishes a vasomotion function toaccommodate pulsatile blood flow, while retaining the luminal gainachieved during the revascularization procedure; wherein the vesselcompliance of the vascular treatment site is acutely restored to a levelapproaching normal limits. In an aspect, the restoration of vesselcompliance occurs immediately after the revascularization procedure.

A method for maintaining luminal gain of a diseased blood vessel,comprising: increasing a luminal gain of a diseased blood vessel using adilatation device; applying a composition comprising a4-amino-1,8-naphthalimide compound to the diseased blood vessel havingan increased luminal gain; activating a composition with an activatingagent to release polyether functional groups; reinforcing a wall of thediseased blood vessel with the released polyether functional groups;wherein the reinforced wall retains the luminal gain withoutcompromising the vascular compliance of the diseased blood vessel.

EXAMPLES Example 1—Synthesis and Initial Purification of a Compound ofFormula (V)

In a 100 mL round bottom flask, 15 grams of JEFFAMINE® 148(Sigma-Aldrich, St. Louis, Mo.) was combined with 1 gram of4-bromo-1,8-naphthalic anhydride (Sigma-Aldrich, St. Louis, Mo.). Thetemperature was held from about 100 to about 110° C. for about 18 toabout 24 hours, and was constantly stirred. The reaction mixture wascooled to room temperature, combined with 50 mL of ethanol(Pharmco-Aaper, Brookfield, Conn.), and then refrigerated at about 4° C.until crystals precipitated from solution (approximately 48 hours). Thecold solution was then filtered by vacuum filtration, and the product,crystals of the compound of formula (V), were washed with 10 mL of coldethanol. The percent yield after precipitation with ethanol wascalculated to be 25%.

Purification of the crystals involved combining the isolated productwith 30 mL of ethanol and heating the mixture to boiling. When all ofthe crystals were dissolved, the heat was removed and the solutioncooled to room temperature, then refrigerated to about 4° C. overnight,allowing crystals to precipitate from solution. The crystals wereisolated using vacuum filtration, rinsed with 10 mL of cold ethanol, andallowed to air dry. No significant losses were recorded during thisrecrystallization step. The material prepared in this fashion wasapproximately 75% pure naphthalimide dimer, the desired product, withthe impurity profile composed of monomeric analogues.

Example 2—Preparation of the Compound of Formula (V) Standard Solution

The naphthalimide solution was prepared as described in Example 1. A 5.0mg/mL solution was prepared by diluting the compound of formula (V) withphosphate-buffered saline (PBS). With constant stirring, the sample pHwas adjusted to 7.4 by dropwise addition of a 10% (v/v) solution ofacetic acid. The final concentration of the solution was confirmed byspectrophotometric analysis (Ocean Optics, USB4000), in which theabsorbance (440 nm) of a 1:200 dilution of the compound of formula (V)solution in isopropyl alcohol was measured. The observed absorbance ofthis sample was 0.5.

Example 3—Naphthalimide Purity by HPLC

A chromatographic separation was performed on a modular HPLC system witha PDA detector and data analysis package (Varian), and detectionwavelengths of 210, 254, 360, and 440 nm, The analytical separation wasachieved using C₁₈ column (Alltima HP, 5 μm, 4.6×250 nm, Alltech) andgradient elution. The elution solvents consisted of mobile phase A, 0.15(v/v) TFA (aq), and mobile phase B, a 90:10 ACN:water with 0.1% (v/v)TFA. A 1:5 dilution (PBS) of the compound of formula (V) naphthalimidestandard solution from Example 2 was analyzed. Using a 20 μL injectionvolume and a flow rate of 1.0 mL/min the standard solution wasintroduced onto a column that had been pre-equilibrated for 10 minuteswith a 95:5 mobile phase ratio of solvents A and B, respectively.Subsequent elution of the analytes was accomplished using a lineargradient with a 1 minute hold at the beginning to 90% mobile phase B at20 minutes, followed by a 5 minute hold at the end of the gradient.

Example 4—Residual Amine Determination by HPLC Analysis

20 μL of the naphthalimide solution from Example 2 was placed in amicro-centrifuge tube and diluted with 1804 μL of reaction buffer (0.15M NaHCO₃, pH=8.6). The sample was vortex mixed and then 2004 μL of thedabsyl chloride reagent (12.4 mM DABS-CI in acetone) was added. Thesamples were incubated at 70° C. for 15 minutes with intermittent vortexmixing, and then cooled in an ice bath for 5 minutes. Next, 4004 of adilution buffer (50:25:25 (v/v/v) acetonitrile:ethanol:mobile phase A(chromatographic mobile phase)) was added and the sample mixed. Sampleswere then centrifuged at 10,000 rpm (Eppendorf 541R) for 5 minutes andthen analyzed.

The chromatographic separation was performed on a modular HPLC systemwith a PDA detector (436 mm) and data analysis package (Varian). Theanalytical separation was achieved using C₁₈ column (Alltima HP, 5 μm,4.6×250 mm, Alltech) and gradient elution. The elution solventsconsisted for mobile phase A, 96% 9 mM Na₂HPO₄, 4% DMF and 0.1% TEA(aq.), and mobile phase B, 80:20 ACN:water. A 204 injection volume andflow rate of 1.0 mL/min were used with a starting mobile phase ratio of92:8 mobile phase A to B, respectively, a two minute hold, and then agradient to 100% mobile phase B at 40 minutes followed by a 10 minutehold. JEFFAMINE® 148 standards were prepared and used to determinelinearity and limit of detection for the compound of formula (V)standard solution.

Example 5—Isolation and Purification of a Compound of Formula (V) UsingPreparative HPLC

A preparative HPLC approach was used to isolate and specifically purifythe compound of formula (V) dimer from the compound of formula (V)naphthalimide product produced from the ethanol crystallization. TheHPLC method used was based on the purity method outlined above; however,method modifications were made to replace acetonitrile with ethanol andTFA with acetic acid in the mobile phase. The preparative separationutilized a C₁₈ column (X-bridge, 10 μm, 19×250 mm, Waters). The mobilephase consisted of mobile phase A, 0.1% acetic acid (aq), and mobilephase B, 9:1 ethanol:water (0.1% v/v acetic acid). The column waspre-equilibrated at a starting mobile phase of 95% A:5% B for 20minutes, at the start of the gradient was a 2 minute hold followed by alinear gradient to 90% B at 40 minutes and a 10 minute hold. Aninjection volume of 8.5 mL (12 mg/mL purified compound of formula (V))and flow rate of 8.5 mL/minute were used as well as a detectorwavelength of 440 nm. The compound of formula (V) fractions werecollected and analyzed using the analytical purity method presentedabove. Fractions meeting a minimum purity criterion were combined andthe solvent driven off by rotovapping the solution to dryness. Excessacetic acid was removed by repeated (two total) dissolution of theproduct in deionized water followed by rotovapping to dryness. Finallythe product was quantitatively transferred from the round bottom flaskto a freeze drying flask by dissolving the product in a minimal amountof deionized water. This solution was frozen at a temperature of −80° C.for a minimum of 24 hours. This final purification approach effectivelyremoved excess solvent and acetic acid from the product as a dimersolution at the desired pH value of 7.4. The naphthalimide dimersolution has demonstrated excellent stability for periods up to oneyear. The dimer in powder form has shown no evidence of degradation forperiods exceeding one year of storage in a dessicator at roomtemperature.

Example 6—Spectral Characterization of the Naphthalimide Monomer andDimer

Standards of the naphthalimide monomer and dimer were analyzed by ESI-MSanalysis for molecular weight (MW) confirmation. Standards were analyzedon a Waters A-TOF I mass spectrometer. A stock solution of the purifiedsolid was prepared in PBS at a concentration of 2 mg/mL. Serial dilutionwas used to prepare solutions of decreasing concentration until thelimit of detection was reached. Absorbance (λ_(max)) curves weremeasured for the naphthalimide dimer using PBS, DMF, and IPA as thedilution solvent. The extinction coefficient for the naphthalimidemonomer was only determined in IPA.

In view of the foregoing, one of ordinary skill in the art would be ableto synthesize the disclosed monomer, dimer, trimer (star, capped,linear) naphthalimide compounds disclosed herein.

Example 7—Plaque Penetration

Diseased arteries were obtained from limb amputations (leg) due toadvanced Periperal Arterial Disease (PAD) from patients at AveraMcKennan hospital. The received arteries were evaluated and chosen suchthat the diseased section was of reasonable size to accommodate thetreatment catheter balloon length being used. The catheter balloondiameter used was also matched to the diameter of the artery and capableof achieving a 1:1.25 (ratio of artery diameter to balloon diameter)overstretch. The artery was laid in a petri dish and an angioplastyballoon was inserted and inflated for 60 seconds to impart the desiredoverstretch. While the angioplasty balloon was inflated a permanentmarker was used to define the treatment zone (where the overstretch wasimparted by the balloon) on the outer surface of the artery. Theangioplasty balloon was deflated and removed. The inner lumen of thediseased artery was exposed to a plaque softening composition comprisinga compound of formula (V) naphthalimide formulation (2 mg/mL inphosphate buffered saline, pH=7.4) for a period of 5 minutes. For thepurpose of filling the artery, one end was clamped, the artery was heldupright and a syringe was used to dispense the naphthalimide solutionuntil the inner lumen was filled. The open end of the artery was thenclamped and the soaking period of 5 minutes commenced. After the 5minute soaking period, the clamps were removed and a treatment catheterwith a dilatation balloon and capable of housing a light fiber for lightactivation was centered in the treatment zone of the artery as definedby the markings made on the outside of the artery. The treatment balloonwas inflated to a similar diameter as the angioplasty balloon in theprevious step and light activation was imparted using the light fibercontained in the central lumen of the catheter and illuminating throughthe treatment balloon. Light activation involved using a laser (447 nm)and a power level of 625 mW/cm delivered to the treatment zone for aperiod of 60 seconds. After completion of light activation, the laserwas turned off, the treatment zone balloon was deflated and the catheterremoved from the artery. Control arteries having no exposure to theplaque softening compound were treated in an identical manner to thosereceiving the plaque softening compound, however, phosphate bufferedsaline (pH 7.4) was used in place of the plaque softening compound,i.e., the naphthalimide compound. Arteries were cut open lengthwise andthe treated section was evaluated by visual examination.

FIGS. 4a and 4b are photos of an untreated section of popliteal arterythat was fairly healthy, having only a slight plaque formation. FIGS. 4cand d are photos of the same artery after angioplasty with a 25%overstretch. FIGS. 4e and f are photos of the same artery after it hasbeen treated, i.e., soaked with the plaque softening compositioncomprising the compound of formula (V).

FIGS. 5a and 5b are photos of an untreated section of tibial artery thatwas partially covered in hard or soft plaque. FIGS. 5c and d are photosof the same artery after angioplasty with a 37% overstretch. FIGS. 5eand f are photos of the same artery after it has been treated, i.e.,soaked with the plaque softening composition comprising the compound offormula (V).

FIGS. 6a, 6b, and 6c are photos of an untreated section of poplitealartery that was partially covered in hard or soft plaque. As can be seenin FIG. 6b the artery was cut open lengthwise (no angioplasty). FIGS.6d, 6e and 6f are photos of the same artery after it has been treated,i.e., soaked with the plaque softening composition comprising thecompound of formula (V).

FIG. 7a is a photo of a section of popliteal artery having large areasof soft plaque. FIGS. 7b and 7c are photos of the same artery afterangioplasty with a 25% overstretch. FIGS. 7d and 7e are photos of thesame artery after it has been treated, i.e., soaked with the plaquesoftening composition comprising the compound of formula (V).

Prior to cutting the artery for visual examination samples from thetreated and untreated sections were collected and preserved forhistological examination. This sample preparation involved usingsurgical scissors to cut a section of approximately 2 mm in length andplacing the section into OCT embedding medium in a plastic mold. Thesample was then immediately immersed in liquid nitrogen for a period ofat least 1 hour. After this time, samples were stored in a −80° C.freezer until ready for further analysis. Histological analysis involvedplacing the frozen section into a cryostat and cutting into sections of10 microns in thickness. Individual sections were placed ontopoly-L-lysine coated glass slide, coverslipped from frozen usingCytoseal 60 and viewed under dark field magnification (10×) using aZeiss Axiovert 200 microscope. Additional histological evaluation wasperformed using H&E staining which allowed for better elucidation of thesoft plaque. The crystalline nature of hard plaque is readily visiblewithout staining. Under this magnification the crystalline structure ofthe calcific plaque was readily visible, as shown in FIG. 8 the darkarea between the two lighter colored areas of the media. Specificlandmarks, such as the presence and appearance of both hard (calcified)and soft plaque, were identified. There was a definitive visualobservation of apparent softened plaque in the treated region. There wasa definitive observation of tacking of intimal flaps.

Microscopic Evaluation—In particular, the treated plaque looked and feltsofter and creamier. See FIGS. 9a-b . FIG. 9a is a photo of an isolatedsection of a blood vessel comprising a plaque matrix. The shrivelednature of the artery represents what the untreated section of the arterylooked like after the artery was cut open. FIG. 9b is a photo is theblood vessel of FIG. 9a after it has been subjected to application ofcomposition comprising a plaque-softening compound. The treated sectionis distinguishable from the untreated portion of the artery as it isdistended and smoother. The appearance of the untreated portion (to theright) is very similar to the shriveled nature of the entire artery asshown in FIG. 10a . The treated and untreated sections of artery wereviewed under a microscope. There was less evidence of calcium crystalsin the treated sections.

Example 8—Tissue Immersion

A sample of human tissue from the common and lower femoral artery wasreceived. It was stored in saline and refrigerated until use. Based uponvisual and tactile observations, the arteries were approximately 7-8 mmin diameter and contained evidence of both hard and soft plaque. Thesoft plaque has a yellow fatty appearance that is distinguishable fromthe vessel wall. The hard plaque presents itself as hard pieces ofmaterial that can be pulled (with a forceps) from the soft plaque.Sections of the artery suitable for catheter work were segregated andstored in phosphate buffered saline (PBS), pH=7.4. From the remainingarterial sections, pieces of tissue containing both hard and soft plaquewere removed. These pieces were either placed in a solution of PBS(pH=7.4), or dimeric naphthalimide, such as a compound of formula (V)(which is in PBS, pH=7.4). Prior to immersion in either solution therelative hardness of the plaque was evaluated via manual manipulationwith a forcep. After a 5 minute soak in solution (typical exposuretime), no difference in the samples soaked in PBS was noticed (control).There was a slight softening of the plaque exposed to a compound offormula (V) (inventive example). The samples were then left in theirrespective solutions for an additional 90 minutes. The plaques soakingin the PBS (control) remained hard, whereas the plaques soaking in thecompound of formula (V) (inventive example) were significantly softerand more pliable.

Example 9—Tissue Receiving Only Angioplasty

The tissue was an artery approximately 7.4 mm in diameter. An Ultra-thinSDS 8 mm×30 mm catheter was used to dilate the artery to 7.55 mm(approximately a 2% overstretch). A fissure or a possible dissectionrunning down the length of the tissue sample was observed. See FIG. 10

Example 10—Tissue Receiving a Plaque-Softening Composition Comprising aCompound of Formula (V) and then Angioplasty

A section (6.5 cm in length) of artery was exposed to the compositionfor 5 minutes. An 80 mm catheter was used to impart a 2% overstretch tothe artery. The angioplasty was followed by photoactivation at 1800 mWfor 60 seconds using the same 80 mm catheter with a 60 mm light fibercentered in the catheter. See FIG. 11a . When this artery was opened upthere was no fissure observed as with the previous artery, however,there did appear to be somewhat of a seam which may indicate thephotoactivated repair of a fissure after the plaque is pre-softened andthen dilated. See FIG. 11 b.

Example 11—Tissue Receiving Angioplasty and then a Plaque-SofteningComposition Comprising a Compound of Formula (V)

A catheter with an 80 mm treatment zone balloon was used however thephotoactivating light fiber was only 60 mm in length. During activationit was noted that the light fiber was shifted distally in the catheter.This meant that the distal end of the artery was receivingphotoactivation, however, the proximal end received little or no light.A fissure down the length of the artery was again observed. At theproximal end where there was minimal light activation there did notappear to be repair. Distally down the artery towards adequatephotoactivation there appeared to be a seam indicating repair. At thevery distal end there was a small flap which indicated that full repairmay not have occurred. See FIG. 12.

Example 12—Other Photoactivated Material

Singlet oxygen generating species, such as Rose Bengal, riboflavin, andmethylene blue were tested to determine if they would be effective atarterial compression. No efficacy was observed, as shown in FIG. 13.

Example 13—Attachment and Release of Pharmacological Agent Tethered to aCompound of Interest

A fresh excised porcine artery can be cleaned of excess tissue andrinsed in PBS. The artery wall can undergo angioplasty to simulateclinical injury. The artery can be filled with a 1.0 mg/mL solution of apharmacological agent bound to a compound of interest, such as anaphthalimide compound, and allowed to soak for 5 minutes. A ballooncatheter capable of accommodating a cylindrically illuminating fiber canbe inflated in the artery to expel any extra material. The artery can beirradiated to activate the naphthalimide compound and effect theattachment of the pharmacological agent to the blood vessel wall. Afterirradiation, the balloon will be deflated and removed. Arterial materialoutside of the treatment zone, that material not around the illuminationzone will be removed and discarded. The artery will be rinsed with PBSand then soaked in PBS multiple times in the dark for at least 1 hour toremove any unbound material.

To determine the attachment of the pharmacological agent to the bloodvessel, the artery can be blotted dry and weighed. The artery will behomogenized and warmed to 40° C. in a basic (high pH) solution. Thisbasic treatment will result in the rapid hydrolysis of the bond, e.g.,an ester bond, and the complete release of the pharmacological agent,e.g., Everolimus. The total amount of the tethered pharmacological agentcan be determined by HPLC.

To determine the release (hydrolysis) rate, a section of treated arterycan be blotted dry and weighted. The artery will be placed in PBS andincubated at 37° C. Aliquots of buffer can be removed and analyzed viaHPLC to determine the amount of pharmacological agent released as afunction of time.

Example 14—A Proposed Synthesis of a Capped Naphthalimide Trimer

Dissolve 0.500 gram of 4-chloro-1,8-naphthalic anhydride (2.16 mmoles,mw=232) in 100 ml of anhydrous ethanol. Add 7.3 mg TEA (0.072 mmoles,mw=101). Add 0.316 grams of JEFFAMINE® T-403 (0.718 mmoles, mw=440) andprotect the reaction vessel with a drying tube. (Note: The reactionproduces water, the reaction may be facilitated by the addition of 20grams of dry molecular sieves.) The reaction should be heated to 50° C.The reaction should be monitored by TLC on fluorescence plates. Theproduct should NOT be fluorescent when viewed under blue (450 nm) light.The reaction will be run for up to 168 hours, if the reaction is notcomplete at this time, the solution will be verified to be basic (addTEA as necessary). If basic, the reaction temperature will be increasedin 10° C. step up to reflux. Chromatography was on silica gel may beused to purify the product, the solvent system to be determined by TLC.

Dissolve 0.53 grams (0.5 mmoles, mw=1065.45) of the4-chloro-⅓T403-naphthalimide in 1000 ml of o-dichlorobenzene(bp=178-180° C.). After the maphthalimide is completely dissolved, add0.22 grams of JEFFAMINE® T-403 (0.5 mmoles, mw=440). The reaction willbe refluxed in the dark under nitrogen and the reaction monitored by theincrease in fluorescence and TLC for up to 168 hours. Partially reactedmaterial will be fluorescent but will be not move from the origin onTLC. While the large volume of solvent has been selected to preventformation of higher order aggregates, it will slow the reactionunacceptably. If the reaction is too slow, the volume of solvent may bereduced.

Example 15—Synthesis of 4-Chloro-Butyl Naphthalimide

Dissolve 0.500 gram of 4-chloro-1,8-naphthalic anhydride (2.16 mmoles,mw=232) with 100 ml of anhydrous ethanol in a 250 ml round bottom flask.Add 0.22 g TEA (2.2 mmoles, mw=101). Place a small stir bar in theflask. Add a cooled reflux condenser to the flask. Place the flask in aheating mantel on a stir plate and heat to reflux. The4-chloro-1,8-naphthalic anhydride should dissolve. After the4-chloro-1,8-naphthalic anhydride has dissolved, add 0.16 g butyl amine(2.2 mmoles, mw=73.14) For ease of handling, add 0.2 ml of butyl aminedirectly through the relux condenser. Allow the reaction to reflux for12-24 hours. Allow the reaction mixture to cool to room temperature. Thereaction can sit until your next day in the lab. Determine the extent ofthe reaction by TLC on normal phase TLC with 50:50 hexane:ethyl acetateas the solvent system. The product should NOT be fluorescent when viewedunder blue (450 nm) light.

Example 16—Addition of Trimeric Jeffamine T-403 to the Reaction Mixtureof Example 15

Return the reaction mixture to reflux. Add 0.316 grams of JeffamineT-403 (0.72 mmoles, mw=440) directly through the condenser. Add 0.22 gTEA (2.2 mmoles, mw=101) directly through the condenser. Allow thereaction to reflux for 48-72 hours. Allow the reaction mixture to coolto room temperature. The reaction can sit until your next day in thelab. If crystals or oil forms, collect by filtration or decanting. Ifthere is no apparent product, add 100 ml of water and cool at 4° C. tobring the product out of solution.

Example 17—Characterization of Product from Example 16

Estimate the purity by TLC on normal phase TLC with 50:50 hexane:ethylacetate as the solvent system. The product should be fluorescent whenviewed under blue (450 nm) light.

Estimate the aqueous solubility by adding small aliquots of product to10 ml of PBS with vigorous stirring. When the solution appears to besaturated and in equilibrium with solid product (or oil), continue tostir for 30 minutes. Then allow to settle for 30 minutes and thenmeasure the absorbance at 440 nm. Make serial 1:10 dilutions in PBS ifthe measured absorbance is above 1.

Dilution A₄₄₀ ~Concentration at A₄₄₀ = 1 None 0.027 mM 1:10  0.27 mM1:100  2.7 mM 1:1000 27 mM

${Concentration} = {\frac{A_{440}}{36,000\mspace{14mu} M^{- 1}\mspace{14mu}{cm}^{- 1}}*{Dilution}\mspace{20mu}( {{Wild}\mspace{14mu}{guess}\mspace{14mu}{for}\mspace{14mu} ɛ} )}$

If the product displays reasonable solubility (>0.05 mM) test forefficacy using the gain model.

Example 18—Synthesis of EDR-148 Terminated Head Linked T-403 Trimer

Dissolve 0.500 gram of 4-chloro-1,8-naphthalic anhydride (2.16 mmoles,mw=232) with 100 ml of anhydrous ethanol in a 250 ml round bottom flask.Add 0.44 g TEA (4.4 mmoles, mw=101). Place a small stir bar in theflask. Add a cooled reflux condenser to the flask. Place the flask in aheating mantel on a stir plate and heat to reflux. The4-chloro-1,8-naphthalic anhydride should dissolve. After the4-chloro-1,8-naphthalic anhydride has dissolved, add 0.326 grams ofJEFFAMINE® EDR-148 (2.2 mmoles, mw=148) followed immediately by 0.316grams of JEFFAMINE® T-403 (0.72 mmoles, mw=440) directly through thecondenser. Allow the reaction to reflux for 48 hours. Allow the reactionmixture to cool to room temperature. The reaction can sit until yournext day in the lab. Estimate the purity and extent of the reaction byTLC on normal phase TLC with 98:2 ethanol:ammonium hydroxide as thesolvent system. The product should be fluorescent when viewed under blue(450 nm) light.

Estimate the aqueous solubility by adding small aliquots of product to10 ml of PBS with vigorous stirring. When the solution appears to besaturated and in equilibrium with solid product (or oil), continue tostir for 30 minutes. Then allow to settle for 30 minutes and thenmeasure the absorbance at 440 nm. Make serial 1:10 dilutions in PBS ifthe measured absorbance is above 1.

Example 20

Porcine internal carotid and femoral arteries (mixed breed and sex) wereobtained from a local slaughterhouse (John Morrell, Sioux Falls, S.Dak.) on an as-needed basis. After excision, specimens were placed overice and delivered to the laboratory within an hour of harvest. Arterieswere rinsed with cold water to remove blood, cleaned using a scissorsand forceps to remove adventitia and connective tissue, and then placedin a beaker of cold phosphate buffered saline (1×PBS, 8 g NaCl, 0.2 gKCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄ into one liter of dH2O, and pHadjusted to 7.4). They were then evaluated for usability (i.e., minimalvessel tapering and collaterals) and cut into approximately 4.5 cmsections. Arteries were then stored for a maximum of 12 hours at 4° C.in PBS until use that same day.

All experiments were conducted at room temperature. In each experiment,arteries were classified as Native, Untreated, or Treated. Nativearteries received no additional treatment. These specimens were used toassess arterial quality prior to testing, or to study the native matrix.Untreated arteries received only balloon angioplasty (BA); Treatedarteries received BA followed by photochemical treatment with a4-amino-1,8-naphthalimide. Prior to BA, arteries were sized (luminaldiameter) so that the correct angioplasty balloon size (Maveric, overthe wire, Boston Scientific, Boston, Mass.; Buckling studies, Fox Plus,Abbott, Abbott Park, Ill.) could be chosen to achieve the desiredoverstretch of 25%. “Overstretch” was defined as the percent increase inluminal diameter that the angioplasty balloon imparted compared to theartery's native diameter. The native diameter was calculated by cuttingoff a small portion of the selected artery, cutting the vessel open,gently flattening it between two glass microscope slides, and then usinga digital caliper to measure the circumference (as length) of the lumen.The luminal diameter was then calculated by dividing the circumferenceby Tr. The catheter used to apply BA was then selected by comparison ofthe calculated luminal diameter to manufacturer sizing charts providedwith the BA catheter. When BA was performed on Untreated and Treatedarteries, the catheter balloon was centered in the artery and theballoon was inflated using an indeflator (Guidant, St. Paul, Minn.)following the catheter manufacturer guidelines. The balloon wasmaintained at the maximum inflation for a period of 60 seconds and thendeflated and removed from the artery. Arteries were irrigated with PBSduring treatment to avoid dehydration, and then placed into PBS for15-60 minutes prior to analysis.

After BA, the process for photochemically treating an ex vivo arterybegan by placing the vessel into a specially designed mounting apparatusand filling the lumen with the disclosed composition (2.5 mM). Thedisclosed composition was allowed to diffuse into the artery for a5-minute period; then the catheter was inserted, centered in the BAtreatment zone, and inflated (25% overstretch). With the ballooninflated, light from a monochromatic 457 nm laser (blue light 2 Watt,Laserglow, Toronto, ON; 457 nm; delivered at 450 Mw) was channeledthrough a custom light fiber to the treatment site for 60 seconds. Afterlight activation, the balloon was immediately deflated and removed fromthe artery. Arteries were bathed with PBS during treatment to avoiddehydration and then placed into PBS after treatment to allow forrelaxation and rehydration.

Measurement of Luminal Gain

After the artery had received the appropriate treatment, the luminaldiameter was calculated from direct measurement of the cross-section(circumference) of the arterial lumen as follows: The artery was cutlongitudinally and, using a digital caliper, three measurements of thecross-section were made in both the treated (n=6) and untreated (n=6-8)sections of artery. The three measurements were averaged and luminaldiameter calculated as specified in the previous section. Luminal gainis defined as the percent increase from the pre-BA measurement asfollows:

Increase in Luminal Diameter Post-BA (mm)−Luminal Diameter Pre-BA(mm)/Luminal Diameter Pre-BA (mm)×100.

Data were expressed as a mean±standard error (S.E.) for n number of exvivo arteries. Unpaired Student's t test or multivariate analysis ofvariance was used for statistical analysis as appropriate.

FIG. 14 shows a plot of the data comparing luminal gain values forarteries receiving BA alone (Untreated) and BA plus photochemicaltreatment (Treated) with the disclosed composition comprising a4-amino-1,8-naphthalimide. Treated arteries clearly demonstratedincreased retention of luminal gain over untreated arteries (i.e., thosereceiving BA alone).

Measurement of Global Stretch Ratio, Distensibility, and ComplianceCoeffecients

The compliance and distensibility of the artery was analyzed bycomparing the change in cross-sectional diameter (mm) as a function ofhydrostatic pressure (mmHg) in the vessel. Native, Untreated, andTreated arteries were prepared as described above (n=6/group). Pressurewas applied and monitored using a pressure transducer, and datacollection software (DATAQ). At zero pressure, four artery diametermeasurements were made using a digital caliper to the outside of theartery and marked with a permanent marker for subsequent measurements.For the Untreated and Treated arteries, these measurements were madecentral to the area receiving treatment with the angioplasty balloon.Immediately after taking zero pressure measurements, pressure in thevessel was increased to 50 mm Hg, and the same measurements were madeafter allowing 30 seconds for pressure equilibration. This process wasrepeated at pressures of 75, 100, 125, 150, and 200 mm Hg. During thisprocess the outside of the suspended artery was kept hydrated byfrequent washes with PBS.

A ratio termed the Global Stretch Ratio (GSR) was calculated by dividingthe artery diameter (d) at a given pressure d_(p), by the diameter atzero pressure d_(o). Cross-sectional area is defined as A=πr², withr=d/2 (A, area; r, radius; d, diameter). Cross-sectional compliancecoefficient is defined as ΔA/ΔP (P=hydrostatic pressure). Distensibilitycoefficient is (ΔA/A)/ΔP.

Compliance values represent the slope of the Global Stretch Ratio (GSR)versus hydrostatic pressure curve. The slope of the curve was measuredfor data points at or above 125 mm Hg, which is the inflection point forwhere the curves differ after the 25% balloon stretch. Data wereexpressed as a mean±S.E. for n number of ex vivo arteries. Multivariateanalysis of variance was used for statistical analysis.

Comparison of diameters for each pressure point revealed differences inthe curves above 100-125 mmHg, for balloon stretched arteries. Thesecurves then became quite different and this is reflected in thecompliance and distensibility measurements. The diameters above 100 mmHgrelate directly to the luminal gain imparted by the BA. Therefore, bothCross-sectional Compliance Coefficient and Cross-sectional Distensibiltywas calculated from 125-200 mmHg and presented in FIGS. 15 and 16. Thedifferences between all three treatment groups are statisticallysignificant (p<0.0001). Most importantly, the Treated arteries (FIGS. 15and 16 “NVS”) are different, which reflected a smaller slope and morenon-compliant behavior than the Untreated arteries (FIGS. 15 and 16“BA”). Because the Treated artery also received BA prior tophotoactivation, the change in GSR compliance value provided compellingevidence that the disclosed method restored vessel compliance incomparison to that of the native blood vessel.

Measurement of Buckling

After the artery was measured for length and balloon size, it wassuspended in PBS and secured at a 30% axial stretch and subjected toincreasing internal hydraulic pressures. The artery was initiallypressurized to 200 mmHg and then released to zero. Then the artery wasinflated from 0 to 100, 150, and then 200 mmHg. Lateral displacement andexterior diameter were measured from photos taken in Native arteries.Buckling was generally only observed after 200 mmHg in healthy, Nativearteries. If a Native artery demonstrated any buckling at 100-150 mmHg,it was discarded and not included in the analysis. Then the artery wasover-stretched to 25% by BA as described above and then pressurized to150 and 200 mmHg. Photos were taken at both pressures. Then the arterywas treated with the disclosed composition and light activated asdescribed above. The artery was then inflated to 150 and 200 mmHg for atotal of three times (NVS1, NVS2, NVS3) and photos were taken again atboth 150 and 200 mmHg. Therefore, each artery was subjected to arterialpressures that caused buckling at Native, then BA (Untreated), and thenNVS (Treated) conditions for a total of 6 inflations to 200 mmHg forevery artery. Photos were taken at a perpendicular angle to the buckle.See FIG. 17. If the artery buckled in a different direction to thecamera, the camera was repositioned in order to take the best photos ofthe maximum buckling of that artery. The central lines of the arterieswere determined by calculating the center of the artery at each end ofthe artery near the coupling and then drawing a straight line betweenthese two central coordinates. Then the displacement (“buckling”) of theartery from the central line was measured at its maximal distance in themiddle of the artery. A ruler was placed by each artery for photocomparison and the displacement was given as millimeters from the centerline of the artery. Occasionally a kinked or twisted appearance wasobserved but only arteries that showed a simple curved buckle were usedin the analysis.

The native artery began to show buckling behavior at 200 mmHg and BAcaused this buckling to increase significantly (from 4.9±2.2 to 9.0±2.0mm, P<0.002, vs BA). After treatment with the disclosed composition andlight activation, the buckling was reduced by about 15% in each arteryand subsequent buckling remained stable at that reduced value (to 7.8,7.7, and 7.9, NVS1, NVS2, NVS3, respectively, p<0.02 vs BA). Arteriesthat were damaged by 25% overstretch with BA were partially repairedwith the disclosed process as evident by reduced buckling in the artery.

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification andexamples be considered as exemplary only, with a true scope and spiritof the invention being indicated by the following claims.

1-24. (canceled)
 25. A method of restoring vascular compliance in adiseased blood vessel, comprising: inserting a delivery system into theblood vessel; applying a bolus of a composition comprising a4-amino-1,8-naphthalimide compound to the blood vessel; and activatingthe composition with a sufficient amount of electromagnetic energy torestore the vascular compliance of the blood vessel; wherein the4-amino-1,8-naphthalimide compound comprises a compound of formula (II):

wherein R, R′, and Q are each independently selected from the groupconsisting of straight-chain and branched chain alkyl groups having from2 to 200 carbons, optionally substituted with one or more ether, amideor amine groups.
 26. The method of claim 25, wherein the blood vessel isan artery or a vein.
 27. The method of claim 25, wherein the vascularcompliance of the blood vessel is restored to its native compliancewithin seconds to about 120 days after the activating step.
 28. Themethod of claim 25, wherein the composition further comprises one ormore excipients, buffers, carriers, stabilizers, preservatives, solventsand/or bulking agents.
 29. The method of claim 28, wherein thecomposition further comprises a solvent.
 30. The method of claim 29,wherein the solvent is phosphate buffered saline.
 31. The method ofclaim 29, wherein the solvent comprises dimethylformamide or isopropylalcohol.
 32. The method of claim 29, wherein the1,4-amino-8-napthalimide compound is present in a concentration fromabout 0.01 mg/mL to about 100 mg/mL.
 33. The method of claim 29, whereinthe 1,4-amino-8-napthalimide compound is present in a concentration fromabout 0.1 mg/mL to about 50 mg/mL.
 34. The method of claim 29, whereinthe 1,4-amino-8-napthalimide compound is present in a concentration fromabout 1 mg/mL to about 30 mg/mL.
 35. The method of claim 25, wherein theelectromagnetic energy comprises blue light.
 36. The method of claim 25,wherein the composition further comprises acetate ions.
 37. The methodof claim 30, wherein the composition further comprises acetate ions. 38.The method of claim 31, wherein the composition further comprisesacetate ions.
 39. The method of claim 25, wherein R and R′ are eachCH₂(CH₂OCH₃)₃CH₂NH₂ and Q is CH₂(CH₂OCH₂)₂CH₂.